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Analysis of E2F1 target genes involved in cell cycle and apoptosis

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Analysis of E2F1 target genes involved in cell cycle and apoptosis
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Freeman, Scott N
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Mitosis
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Mcl-1
Rb
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ABSTRACT: One of the main results of Rb-E2F pathway disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the induction of apoptosis. Given the potential negative biological effects associated with deregulated E2F activity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct physiological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregulated during mitosis due in part to E2F1, and that overexpression of RhoBTB2 increases the S-phase fraction and slows the rate of proliferation. We also find RhoBTB2 similarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis.^ Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL-1---MCL-1 +6 and +18---as initially published by Moshynska et al (1). In contrast to Moshynska et al., we find the variant promoters identically present in both cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that observed in cancerous cell lines. In further contrast, we find the activity of the MCL-1 +6 and +18 promoters approximately 50% less than the common MCL-1 +0 promoter---both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription.^ Given our results and those of others, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no sic represent a reliable prognostic marker for CLL as reported by Moshynska et al.
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Dissertation (Ph.D.)--University of South Florida, 2007.
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Includes bibliographical references.
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by Scott N. Freeman.
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Analysis of E2F1 Target Genes Involved in Cell Cycle and Apoptosis by Scott N. Freeman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cancer Biology College of Graduate Studies University of South Florida Major Professor: W. Douglas Cress, Ph.D. Srikumar P. Chellappan, Ph.D. Eric B. Haura, M.D. Kenneth L. Wright, Ph.D. Date of Approval: October 15, 2007 Keywords: Cancer, Mitosis, Transcription, Mcl-1, Rb Copyright 2007, Scott N. Freeman

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DEDICATION To my parents Thomas N. and Linda L. Fr eeman, my brother Michael T. Freeman, and my future wife Alyson K. Fay.

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ACKNOWLEDGEMENTS I would like to thank my di ssertation advisor and mentor W. Douglas Cress, Ph.D. for the years of guidance, assistance, and trai ning that he contributed to and invested in my development as a scientist, my dissertation committee members Srikumar P. Chellappan, Ph.D., Eric B. Haura, M.D., and Kenneth L. Wright, Ph.D. for their guidance and direction throughout my doctoral training, David G. Johnson, Ph.D. for being so kind as to serve as my outside chair, Eric B. Haura M.D., Rebecca Sutphen, Ph.D, Yihong Ma, Ph.D., and Gerold Bepler, M.D, Ph.D., and the core facilities of the H. Lee Moffitt Cancer Center and Research Institute for their respective contributions to this manuscript, and finally the Cancer Biology Ph.D. Program, the University of S outh Florida, and the H. Lee Moffitt Cancer Center and Research Center for providing me with the opportunity to accomplish this goal.

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NOTE TO READER The original of this document contains colo r that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida

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i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF ABBREVIATIONS vii ABSTRACT ix PART I: RHOBTB2 (DBC2) IS A MI TOTIC E2F1 TARGET WITH A NOVEL ROLE IN APOPTOSIS 1 Abstract 2 Introduction 3 The Rb-E2F Pathway 3 Mechanisms of Rb-E2F Pathway Disruption in Human Malignancy 6 Deregulated E2F Activity 9 The E2F Family of Transcription Factors 10 Promotion of Proliferation and Oncogenesis 13 Promotion of Apoptosis 15 Contradictory Roles: Promoti on of Growth Arrest, Tumor Suppression, and Survival 17 E2F Target Genes: Connecting th e Biology of Deregulated E2F to Mechanisms 19 Mitotic Targets of E2F 20 Apoptotic E2F Targets and Mechanisms 21 E2F Targets and Mechanisms Involved in Growth Arrest, Tumor Suppression, and Survival 25 The RhoBTB2 (DBC2) Putative Tumor Suppressor Gene 26 Structure 26 Expression Patterns 27 Deregulation in Human Malignancy 28 Biological Functions, Mechanisms, and Regulation 28 Summary and Rationale 30 Experimental Procedures 32 Cell Lines and Cell Culture 32 Adenovirus 32 Real-Time PCR 33 RhoBTB2 Antibody Production 34 Plasmids, siRNA, and Transfections 34

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ii Immunofluorescent Microscopy 35 Flow Cytometry 35 MTS Assays 36 Results 37 E2F1 Overexpression Upregulates RhoBTB2 37 Upregulation of RhoBTB 2 by E2F1 is Direct and not Dependent on Artificial Overexpression 41 RhoBTB2 is Upregulated During Mitosis, which is Partially Dependent on E2F1 43 Overexpression of RhoBTB2 Increases the S-phase Fraction and Slows Proliferation 47 RhoBTB2 is Upregulated During Dr ug-Induced Apoptosis, which is Primarily Dependent on E2F1 49 Knockdown of RhoBTB2 Expressi on by siRNA Impairs the Induction of Drug-Induced Apoptosis 53 Discussion 55 PART II: IDENTIFICA TION AND CHARACTERIZATION OF TWO NOVEL MCL-1 PROMOTER POLYMORPHISMS 59 Abstract 60 Introduction 62 Mcl-1 and the Bcl-2 Family of Proteins 62 The BH3-Only Subfamily 64 The Bcl-2 Subfamily 64 The Bax Subfamily 65 Mcl-1 is an Inhibitor of Apoptosis 66 Mcl-1 and Oncogenic Transformation 68 Mechanisms Regulating Mcl-1 Expression 68 Mcl-1 and Human Malignancy 72 Summary and Rationale 73 Experimental Procedures 75 Promoter Identification and Screening 75 Cell Lines 76 Paired Clinical Lung Samples 76 Healthy Control Samples 76 Luciferase Assays 77 Results 78 Identification of Two Novel MC L-1 Promoter Variants 78 The MCL-1 +6 and MCL-1 +18 Promoter Variants are not the Result of Somatic Mutation 79 The MCL-1 +6 and MCL-1 +18 Promoters are Common Polymorphisms 79 The MCL-1 +6 and MCL-1 +18 Promoters are Less Active than the Common MCL-1 +0 Promoter 82 Discussion 84

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iii LIST OF REFERENCES 87 ABOUT THE AUTHOR END PAGE

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iv LIST OF TABLES Table 1 The allelic frequencies of the MCL-1 +0, MCL-1 +6, and MCL-1 +18 promoters in breast a nd lung cancer cell lines. 80

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v LIST OF FIGURES Figure 1. The Rb-E2F pathway 5 Figure 2. Examples Rb-E2F pathway di sruptions in human malignancy 7 Figure 3. The E2F family of transcription factors 11 Figure 4. Mechanisms of E2F1-induced apoptosis 22 Figure 5. E2F1 overexpression up regulates RhoBTB2 mRNA 38 Figure 6. Novel RhoBTB2 antibody is functional in immunofluorescent microscopy and is specific for RhoBTB2 40 Figure 7. E2F1 overexpression upre gulates RhoBTB2 protein 42 Figure 8. E2F1-mediated upregulati on of RhoBTB2 is direct 42 Figure 9. RhoBTB2 is a physiolo gical target of E2F1 44 Figure 10. RhoBTB2 is upregulated during mitosis 46 Figure 11. Mitotic upregulat ion of RhoBTB2 is partially dependent on E2F1 46 Figure 12. Overexpression of RhoBTB2 increases the S-phase fraction and slows proliferation 48 Figure 13. RhoBTB2 is upregulated during drug-induced apoptosis 50 Figure 14. Upregulation of RhoBTB2 during drug-induced apoptosis is primarily dependent on E2F1 52 Figure 15. Knockdown of RhoBTB2 via si RNA impairs the induction of druginduced apoptosis 54 Figure 16. A proposed mechanistic model for RhoBTB2 activity 58 Figure 17. The Bcl-2 family and the intrinsic stress-i nduced apoptotic pathway 63

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vi Figure 18. Mechanisms regulating Mcl-1 69 Figure 19. The variant MC L-1 promoters are not th e result of somatic mutation 80 Figure 20. The variant MC L-1 promoters are prevalent in genomic DNA derived from healthy controls 81 Figure 21. The MCL-1 +6 and MCL-1 +18 promoters are less active than the MCL-1 +0 promoter 84

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vii LIST OF ABBREVIATIONS 4-OHT 4-hydroxytamoxifen ALL Acute lymphoblastic leukemia Apaf-1 Apoptosis activating factor-1 ATM Ataxia telangiectasia mutated Bcl-2 B-cell lymphoma/leukemia-2 BH1-4 Bcl-2 homology 1-4 BTB/POZ Broad-complex bric -a-brac/poxvirus zinc finger Cdc2 Cell division control 2 Cdk Cyclin-dependent kinase ChIP Chromatin immunoprecipitation CHX Cyclohexamide CKI Cyclin-dependent kinase inhibitor CLL Chronic lymphocytic leukemia CREB cAMP response element-binding CRE cAMP response element Cul3 Cullin 3 DBC2 Deleted in breast cancer 2 DP DRTF1-polypeptide E2F Early 2 factor

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viii ER Estrogen receptor FBS Fetal bovine serum GFP Green fluorescent protein hTERT Human telomerase reverse transcriptase IFM Immunofluorescenct microscopy IRES Internal ribosome entry site K5 Keratin 5 Mcl-1 Myeloid cell leukemia-1 Mdm2 Murine double minute 2 NES Nuclear exclusion sequence NLS Nuclear localization sequence PCR Polymerase chain reaction PI Propidium iodide PMA Phorbol 12-myristate 13-acetate P/S Penicillin/streptomycin Rb Retinblastoma RYBP RING1 and YY1 -binding protein shRNA Short hairpin inhibitory RNA SIE Serum-inducible element siRNA Small inhibitory RNA Skp2 S-phase kinase-associated protein 2 TM Transmembrane TRAF2 TNF Receptor-Associated Factor 2

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ix ANALYSIS OF E2F1 TARGET GENES INVOLVED IN CELL CYCLE AND APOPTOSIS Scott N. Freeman ABSTRACT One of the main results of Rb-E2F path way disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the inducti on of apoptosis. Given the pot ential negative biological effects associated with deregulated E2F activ ity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct phys iological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregul ated during mitosis due in part to E2F1, and that overexpression of R hoBTB2 increases the S-phase fr action and slows the rate of proliferation. We also find RhoBTB2 si milarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis. Taken together we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL1MCL-1 +6 and +18as initially published by Moshynska et al (1). In c ontrast to Moshynska et al., we find the variant promoters identically present in both cancerous and ad jacent noncancerous clinical lung samples,

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x suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samp les and present at frequencies similar to that observed in cancer ous cell lines. In furthe r contrast, we find the activity of the MCL-1 +6 and +18 pr omoters approximately 50% less than the common MCL-1 +0 promoterboth during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription. Given our results and those of others, we conclude that the MCL-1 +6 and +18 pr omoters are likely benign polymorphisms and do no represent a reliable prognostic marker for CLL as reported by Moshynska et al.

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1 PART I RHOBTB2 (DBC2) IS A MITOTIC E2F1 TARGET WITH A NOVEL ROLE IN APOPTOSIS

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2 Abstract We have identified the RhoBTB2 putativ e tumor suppressor gene as a direct target of the E2F1 transcription factor. Over expression of E2F1 lead s to upregulation of RhoBTB2 at the levels of mRNA and protein. Th is also occurs during the induction of an estrogen receptor-fused E2F1 construct by 4-hydroxytamoxifen in the presence of cyclohexamide, thus indicating that RhoB TB2 is a direct target. RNAi-mediated knockdown of E2F1 expression decreases RhoBTB2 protein expression, demonstrating that RhoBTB2 is a physiological target of E2F1. Since E2F1 primarily serves to transcribe genes involved in cell cycle pr ogression and apoptosis, we explored whether RhoBTB2 played roles in either of these processes. We find RhoBTB2 expression highly upregulated during mitosis, which is part ially dependent on the presence of E2F1. Furthermore, overexpression of RhoBTB2 leads to an increase in the S-phase fraction of asynchronously growing cells and also slows the rate of proliferation. We similarly find RhoBTB2 upregulated during drug-induced a poptosis, and that this is primarily dependent on E2F1. Finally, we demonstrate that knockdown of RhoBTB2 levels via siRNA slows the rate of druginduced apoptosis. Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis.

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3 Introduction The Rb-E2F pathway The Retinoblastoma (Rb)-Early 2 Factor (E 2F) pathway is a critical regulator of molecular mechanisms governing various aspe cts of cell prolifer ation, differentiation, and survival (for review, see refs. (2-6)). It regulates these biological effects by integrating both positive and negative signals to ultimately control the transcriptional repression or activation of genes involved in the aforementioned processes. Given the importance of tight regulation of proliferation, differentiation, and survival in the avoidance of human malignancy, it is not surpri sing to find that this pathway is aberrantly regulated by various means in almost ever y instance of human malignancy (7). One of the results of deregulation of the Rb-E2F pathway is unrestrained transcriptional activation by certain members of the E2F fam ily of proteins, which can contribute to oncogenic transformation (4). Indeed, many iden tified E2F target genes play direct roles in the biological effects associated with deregulation of the Rb-E2F pathway (8,9). Yet while many crucial E2F targets associated wi th the biological phe notype of deregulated Rb-E2F have been identified, many more remain to be characterized. Given the prevalence of Rb-E2F pathway deregulation in human malignancy and the role of E2F

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4 targets in mediating the biological effects, characterizing E2F target genes involved in this process is of great importance. At the center of a cells decision to divide is the Rb-E2F pathway, and as such one of its major roles is to regulate the G1/S-phase transition. While the function of the pathway encompasses more than regulation of this transition, its model of activity is best explained under that context. In this model, the Rb-E2F pa thway responds to both proand anti-proliferative signals to either activ ate or repress the tr anscription of genes involved in further cell cycle progressi on and DNA synthesis. Many reviews have thoroughly documented this functional paradigm (6,10,11), and the reader is encouraged to reference these for greater detail. As su ch, only a brief description of the current paradigm is provided. As illustrated in figure 1, in cells that ar e in a resting or quiescent state, the pRb protein resides hypophosphorylated, which allows it to restrain the transcriptional activity of E2F proteins. Mitogeni c signaling in early G1 or G0 serves to upregulate the expression of D-type cyclinsthe regulatory subunit of th e cyclin D/cyclin-dep endent kinase (cdk) 4/6 complex. Cyclin D binds to cdk4/6 to create the active kinase complex, which along with the reported activity of Raf-1, places the initial phosphorylation events on pRb family proteins (12-14). Phos phorylation of pRb family proteins decreases their ability to inhibit E2F family members, thus freeing some transcriptionally active DRTF1polypeptide (DP)/E2F complex. This free co mplex sets in motion a feed-forward mechanism that results in increased expression of E2F target genes such as E2F1, E2F2, and E2F3a, as well as cyclin E, the regulat ory subunit of the cyclin E/cdk2 complex

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E2F pRB DP Mitogens Cyclin D Cyclin D Cdk4/6 Cdk4/6 Cyclin D Cdk4/6 Cyclin D Cdk4/6 Cyclin E Cyclin E Cdk2 Cdk2 Cdk2 E2F DP1 E2F DP1 Inactivep E2F pRB DP pRB p p p p pRB p p p p G0/G1G1/S Transcription off Transcription on Cyclin A Cdk2 E2F DP p p p p Cyclin E Cyclin E DNA pol DHFR Raf-1 Raf-1 S Inactive Figure 1. The Rb-E2F pathway. Mitogenic signaling in G0/G1 upregulates cyclin D1 and Raf-1, which contributes to phosphoryla tion of pRb family pr oteinsthus relieving some inhibition of E2F/DP complex. Further E2F-mediated upregulation of cyclin E at the G1/S-phase transition leads to additional phosphorylation of pRb by cyclin E/cdk2 complex, leading to full inactivating of pRb. This initiates S-phase entry and allows E2F/DP complex to activate the transcripti on of genes involved in DNA replication, further cell cycle progression, and genes th at subsequently deactivate E2F and DP. 5

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6 (15-18). Cyclin E binds to cdk2, the catalytic subunit, to crea te the active kinase complex, and the main target of the cyclin E/cdk2 co mplex is again the pRb protein (19). Cyclin E/cdk2 complex fully phosphorylates pRb, t hus allowing for the full induction of E2F target genes. Among the many genes induced by E2F family proteins at this stage of the cell cycle include those involved in further cell cycle progression, DNA replication, and nucleotide biosynthesis. Subseque nt sections discuss the full ra nge of E2F target genes in greater detail; however, it shoul d be noted that two important targets of E2F at this stage of the cell cycle are cyclin A and S-phase ki nase-associated protein 2 (Skp2), which are responsible for down-regulati ng E2F activity through two separate mechanisms (20,21). Cyclin A is another regulato ry subunit for cdk2 which, along with promoting further cell cycle progression, phosphorylates E2F and DP family proteins when in complex with cdk2resulting in a decreased ability to bind DNA (19,22). Skp2 activity also decreases E2F activity through ubquitination, thus targeting it for proteasomal degradation (23). Mechanisms of Rb-E2F pathway disruption in human malignancy One of the defining features of malignanc y is uncontrolled cellular proliferation, and given the pivotal role that the Rb-E2F pathway plays in regulating this process, it is not surprising to find that disruption of th e Rb-E2F pathway is a unifying factor in virtually every instance of human malignancy (7). An examination of figure 1 reveals multiple potential points for deregulation, and indeed, most have been described in the literature. Figure 2 provides examples of va rious methods employed by malignant cells to

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Mitogens Cyclin D/Cdk4/6 Rb-p Rb E2F S phase CKIActivating mutations in RTKs Activating mutations in Src Amplification/upregulation of Cyclin D1 and Cdk4 Deletion of p16INK4aDeletion or mutation of Rb Amplification of E2F3 7 Figure 2. Examples of Rb-E2F pathway disruptions in human malignancy. The RbE2F pathway is subject to va rious regulatory mechanisms that prevent inappropriate proliferation, however malignant cells overr ide these controls through various oncogenic mutations. Some examples described in hum an malignancy include activating mutations in receptor tyrosine kinases, activating mutations in signa ling molecules such as Src, amplification or upregulation of cy clin D or cdk4, deletion of CKI p16INK4a, deletion or mutation of Rb, and amplification of E2F.

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8 circumvent control mechanisms preventing inappr opriate entry into the cell cycle, yet this is by no means a comprehensive list of all re ported mechanism utilized to deregulate RbE2F pathway in human malignancy. The most obvious and most prominent point of deregulation lies with the RB1 gene itself. Indeed, the RB1 gene was first described in its namesake retinoblastoma as being the inherited genetic component behi nd this childhood familial malignancy of the eye (24,25). Interestingly, RB1 has the notorious distinction of being the first identified tumor suppressor gene. While identified as an inherited genetic component contributing to malignancy, it has become clear that somatically arising disruptions of the RB1 gene by means of deletion or muta tion are more common in malignancy than inherited germline mutations (7). In addition to the RB1 gene itself, genetic altera tions in regulators of pRb phosphorylation status are also very prevalent. The p16INK4A protein is a member of a family of cyclin-dependent kinase inhibito rs (CKIs) that directly oppose the action of cyclin/cdk complexes. p16INK4A specifically inhibits the activity of cyclin D/cdk4/6 complexes, thus inhibiting pRb phosphoryla tion (26). Not withstanding, disruption of p16INK4A activity by means of deletion, mutation, or promoter methylation is also well documented. Similarly, the p16INK4A target cyclin D/cdk4/6 is fr equently altered in cancer by means of amplification or tr anslocation of either cyclin D or cdk4/6. The end result of both of these aberrances is unwarranted inactivation of pRb (7). It was long thought that genetic aberran ces in E2F genes themselves were not a common occurrence in malignancy, yet recent reports have identified a handful of genetic alterations in E2F. Amplification of E2F3 is present in some retinoblastomas and urinary

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9 bladder carcinomas (27-31), and amplification of E2F1 has been reported in melanoma, colorectal, esophageal, and ovarian cancers ( 32-37). While upregulation of the activating E2Fs is a common occurrence in malignancy, it is not understood why genetic aberrances in the E2F gene itself are not more prevalent. Deregulated E2F activity One of the main results of Rb-E2F path way disruption is deregulation of the E2F family of transcription factors. This can mani fest itself through both the loss of ability to repress E2F target genesmediated primarily by the repressive E2Fs in complex with pRb, and the loss of ability to restrain gene transactivation, which is primarily a function of the activating E2Fs. Since the subsequent experiments concentrate on the consequences of deregulated E2F-mediat ed gene transactivation in malignancy, mechanisms relating to the loss of ability to repress E2F target genes are not discussed. Likewise, studies utilizing loss-of-functi on techniques to determine physiological functions of E2F are also not discussed. Inst ead, the subsequent sections describe the various members and subgroups within the E2F family and the biological effects associated with deregulated E2F transac tivationprimarily being the promotion of proliferation and oncogenesis and the inducti on of apoptosis. It should be noted that under some contexts, deregulated E2F can para doxically promote surv ival, induce growth arrest, or contribute to tumor suppression (38-45). While the mechanisms and contexts of these biological effects are not as well-defin ed, in many instances they are dependent on the presence of one or more tumor suppressors such as p19ARF, p53, p21, p16INK4A, or Rb,

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10 which is often not the case in cancer (42-45). However, studi es examining this seemingly contradictory role of deregulated E2F are relevant to the pres ent study and are also addressed. The E2F family of transcription factors Nine E2F family members have been id entified to date (E2F1-8, with E2F3 having two variants: E2F3a and E2F3b) and ha ve traditionally been divided into three subgroups based on both structure and func tion (46-62). However, emerging data illustrating the highly complex nature of function within the E2F family has rendered this view overly simplistic (3). It is clear though that in genera l terms, certain subgroups of E2Fs are more associated with either target gene transactivation or target gene repression, and in the interest of presenting an overvi ew of members within the E2F family, the traditional model will be utilized. E2F1, E2F2, and E2F3a constitute the first subgroup of E2Fs and are commonly referred to as the activating E2Fs by virt ue of their ability to potently activate the transcription of genes from model promoters. Structurall y, these E2Fs contain an Nterminal nuclear localization sequence (NLS) and cyclin A/cdk2-binding domain followed by a DNA-binding domain, a DP dimerization domain and a C-terminal transactivation/pRb-binding domain (Fig. 3, t op). These E2Fs associate exclusively with pRb and not p107 or p130. In normal cells, the expression of these E2Fs is tightly coupled to cell cycle, with expression increasing transcriptionally upon mitogenic stimulation in G1 (15,16,63), and decreasing in part due to post-translational modification

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NLS and Cyclin A/Cdk2-binding DNAbinding NES DP Dimerization pRb family binding E2F1 E2F2 E2F3aActivatingE2F3b E2F4 E2F5Repressive E2F6 E2F7 E2F8Atypical RYBPbinding Figure 3. The E2F family of transcription factors. The E2F family of transcription factors is commonly divided into the activating, repressi ve and atypical subgroups. The activating E2Fs consist of E2F1, E2F2, and E2F3a and contain a NLS and cyclin A/cdk2 binding domain, DNA-binding domain, DP dimerization domain, and a pRb family member-binding domain. The repressive E2Fs are E2F3b, E2F4, and E2F5 and contain a DNA-binding domain, DP-dimerization domain, and pRb family member-binding domain. While E2F3b contains a NLS, E2F4 and E2F5 harbor a NES. The atypical E2Fs are E2F6, E2F7, and E2F8. E2F6 contains a DNA-binding domain and RYBP-binding domain, and E2F7 and E2F8 contain a tandem of two DNA-binding domains. 11

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12 imposed by the activity of Skp2 in late Sphase, which targets E2F1 for proteasomal degradation (23). These E2Fs have been imp licated in promoting the transcription of a multitude of genes with various cellular function s, which is discussed in greater detail in further sections. The second subgroup of E2Fs is made up of E2F3b, E2F4, and E2F5 and is commonly referred to as the repressive E2 Fs due to their poor ability to activate transcription, as well as their potent ability to repress transcription when in complex with pRb family members. While E2F3b contains an N-terminal NLS and cyclin A/cdk2binding domain, this sequence is absent from E2F4 and E2F5, which instead have a nuclear exclusion sequence (NES) following the DNA-binding domain (Fig. 3, middle). These E2Fs also contain a DP dimeriza tion domain and a C-terminal pRb family member-binding domain. While E2F3b associates exclusively to pRb, E2F4 can associate with pRb, p107, or p130, and E2F5 only associ ates with p130. In contrast to the activating E2Fs, expression of the repressive E2Fs is relatively st atic throughout the cell cycle. Given the constant nature of expressi on of E2F4 and E2F5, it stands to reason that other mechanisms are in place to regulate th eir activity. Indeed, these E2Fs are regulated by localizationwith inactive E2F4 and E2F5 being cytoplasmic and association with pRb or DP family members being required fo r nuclear import (11). It appears that the primary role of E2F3b, E2F4, and E2F5 is to repress the transcription of E2F target genes through the recruitment of repressive co mplexes containing pRb family members. The final subgroup of E2Fs will be referred to as the atypical E2Fs due to their divergence from E2F1-5. These E2Fs have b een identified more recently, and therefore less is known about their cellular functions. E2F6 was the first id entified atypical E2F

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13 and contains only one E2F family conserve d sequence: the DNA-binding domain (Fig. 3, bottom). Given the absence of a pRb-binding do main, E2F6 does not bind to pRb family members, but instead recruits component s of the mammalian polycomb group complex through a RING1 and YY1-binding protein (RYBP) domain to repress the transcription of E2F target genes (64). E2F7 and E2F8 repr esent an entirely new class of E2Fs whose homology to other E2F family members is limited to a tandem of two DNA-binding domains (Fig. 3, bottom). Given the lack of pRb-binding or dimerization domains, these E2Fs are thought to bind DNA independent of DP or pRb. The limited amount of studies examining the functions of E2F7 and E2F8 suggest that these proteins act as repressors of transcription through as yet uncha racterized mechanisms (58,59,62,65). Promotion of proliferation and oncogenesis One of the most pronounced biological e ffects of unrestraine d transactivation by the activating E2Fs is the promotion of cell cycle progression, which is typically manifested as inappropriate S-phase entry. In cell culture-based assa ys utilizing rodent fibroblasts, overexpression of E2F1, E2F2 or E2F3a is capable of inducing S-phase entry from quiescence (66-69), and in the case of E2 F1, can override anti-proliferation signals imposed by the expression of CKIs p16, p21, p27 or treatment with TGF(70-72). This potent ability to promote cell cycle progression can also manifest in the transformation of primary cells, where overexpression of E2F1, E2F2, or E2F3 can induce transformation either alone or in combination with oncogenic ras (73-76).

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14 The pro-proliferative and oncogenic eff ects of E2F overexpression observed in cell culture-based assays are also evident in vivo by means of mice transgenic for E2F, where transgenic expression of E2F can promot e inappropriate entry into the cell cycle, hyperplasia, and even tumor fo rmation. Consistent with the in vitro models, transgenic expression of E2F1, E2F2 or E2F3a targeted to the lens fiber is capable of inducing reentry into the cell cycle in postmitotic cells (77,78). Transgenic E2F4 can also induce cell cycle reentry in this model, albeit to a lesser extent (77). When expressed under control of the megakaryocyte-specific platelet factor 4 promoter, E2F1 blocks terminal differentiation and induces prol iferation in megakaryocytes, and the differentiation block imposed cannot be rescued by administrati on of platelet growth factors (79). Furthermore, short-term induction of an E2F3 transgene in the p ituitary gland induces proliferation of quiescent melanotrophs (45) indicating that long-term expression of deregulated E2F is not necessary to obs erve a biologically relevant effect. While short-term induction of E2F3 in the pituitary gland induces the proliferation of quiescent cells, long-term induction leads to the development of hyperplasia (45), and targeting transgenic expression of E2F1 or E2F3a to the epidermis and squamous epithelial tissues via the keratin 5 (K5) promoter also results in hyperplasia (80,81). Similarly, targeting transgenic E2F2 to the thymic epithelium results in hyperplasia (82). When targeted to the liver, transgenic E2F1 leads to pericentral large cell dysplasia (83), and conditional expression of E2F1 in the testes from an inducible promoter induces dyplasia that mimics carcinoma in situ indicating that short-term E2F expression is sufficient to drive aberrant tissue proliferation in vivo (84).

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15 In addition to the promotion of aberra nt non-malignant tissue proliferation, transgenic expression of E2F can also lead to tumor developmenteither alone or in combination with other oncogenic mu tations. In the presence of oncogenic ras or the absence of one or both p53 alleles, transgenic expression of E2F1 in K5 tissues leads to the development of skin tumors (80,85). Furthe rmore, K5 E2F1 transgenic mice are also prone to the spontaneous development of tumors in K5-expressing tissues as they age (40). In addition to dysplasia, transgenic e xpression of E2F1 in the liver also induces spontaneous tumor development (83), and targ eting of E2F2 to the thymus epithelium can similarly induce tumor development in a ddition to hyperplasia (82). In the case of E2F3a, transgenic expression to K5 tissues increases the rate of spontaneous tumor development by 20% and additionally enhan ces tumor development in response to treatment with chemical carcinogens (81). Ta ken together, these st udies demonstrate the ability in vitro and in vivo of deregulated E2F activity to promote cell proliferation in presence of antiproliferative signals, promote aberrant non-m alignant tissue growth, and in some contexts, to promote tumorigenesis alone or in combination with other oncogenic mutations. Promotion of apoptosis In addition to promoting cell cycle progression and oncogenic transformation, E2F1, E2F2 and E2F3a also have the abil ity to induce apoptosis, although there is significant disagreement as to the apoptosis-inducing ability of E2F2 and E2F3a (45,68,77,78,81,82,86,87). This is thought to act as a failsafe mechanism to counteract

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16 the potential tumorigenicity associated with unrestrained E2F-mediated proliferation, and can occur in both p53 family-dependent a nd independent mechanisms, which are described in detail in the s ection on E2F1 target genes. In cell culture-based experiments, ectopi c overexpression of E2F1 in quiescent rodent fibroblasts by means of cDNA or ad enovirus results in both S-phase entry and apoptosis (67,74,88,89). While the ability of E2F1 to induce apoptosis in vitro is quite clear, cell culture-based studies examining a role of E2F2 and E2F3a in E2F-induced apoptosis have yielded conflicting results While one study reports no increase in apoptosis upon E2F2 or E2F3a overexpression (68), others have reported the contrary (86,87). Given this apparent cont radiction, it is likely that the ability of E2F2 and E2F3a to induce apoptosis is highly context-dependent, whereas the ability of E2F1 is more ubiquitous. The ability of E2F overexpression to indu ce apoptosis as observed in cell culturebased assays is also evident in vivo by means of mice transgenic for E2F. In addition to E2F1 blocking differentiation a nd inducing proliferation when transgenicly targeted to megacaryocytes, significant megakaryocyte a poptosis is also observed (79). Likewise, when targeted to the liver or lens fiber, tr ansgenic E2F1 induces pr oliferation as well as apoptosis (78), and an inducible E2F1 transg ene targeted to the testes also promotes proliferation and apoptosis (84)indicating that short-term deregulation of E2F1 is sufficient to drive apoptosis in vivo Targeting of E2F1 to the K5 expressing epidermal tissues induces follicular apoptos is, and when crossed to a p53+/or p53-/background, E2F1-induced keratinocyte apoptosis is reduced (85)indicating a ro le for the p53 tumor suppressor gene in E2F1-induced apoptosis. Oddly, when expressed under a non tissue-

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17 specific promoter, transgenic expression is on ly observed in the testicles and results in atrophy and sterility by means of increased apoptosis in the germinal epithelium (90). This however is independent of p53, as crossing these mice to a p53-/+ or p53-/background does not result in decreased apoptosis (90). Similar results have also been obtained in mice transgenic for E2F2 or E2F3a. Trangenic expresion of E2F2 or E2F3a in the lens fiber promotes cel l cycle reentry with subsequent apoptosis in postmitotic cells ( 77,78), yet there is no evident increase in apoptosis when E2F2 is targeted to the thymic epithelium (82), or when E2F3a is targeted to the pituitary gland (45). However, targeted expression of E2F3a to K5 tissues results in increased p53-independent apoptosis, as i ndicated by no decrease in the proportion of apoptotic cells when crossed to a p53-null background (81). As with in vitro-based studies examining a role for E2F2 and E2F3a in apoptosis induction, it is likely that their ability to induce apoptosis in vivo is also highly context depe ndent. It should also be noted that a recent study demonstrates that a poptosis induced by transgenic expression of E2F3a is dependent on E2F1 (91). In summary, under some contexts deregulated E2F, primarily E2F1, is capable of promoting a poptosis in addition to cell cycle progression though both p53-dependent and independent pathways. Contradictory roles: promotion of growth arrest, tumor suppression, and survival The previous sections discuss the ability of deregulated E2F to promote cell cycle progression, apoptosis, and oncogenesis, howev er it should be noted that under some contexts deregulated E2F can promote somewhat contradictory biologi cal effects such as

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18 growth arrest, tumor suppression, and cell surviv al (38-45). In cell culture-based assays, overexpression of E2F1 in primary fibroblasts can induce a growth arrest or checkpoint response that in some instances resembles a senescent-like state, which is dependent on the presence of one or more poten t tumor suppressor genes such as p19ARF, p53, p21, p16INK4A, or Rb (42-45). Similarly, when transgenically targeted to the pituitary gland, E2F3 can induce an irreversible senescen t-like state upon long-term exposure (45). However, reports of E2F-mediated growth arrest are sparse, and under most published contexts deregulated E2F induces proliferation. In the case of tumor suppression, overe xpression of E2F1 in transformed mouse fibroblasts or normal human foreskin fibrobl asts can reduce colony formation, and in the context of mouse fibroblasts, can abrogate focus formation induced by ras (38,39). The necessity of a functional tumor suppressor in this process is exemplified by the ability of dominant-negative p53 to abrogate the ability of E2F1 to suppress focus formation (39). While transgenic expression of E2F1 in K5 expressing tissues can lead to hyperplasia and the development of spontaneous tumors, it paradoxically suppre sses tumor formation induced by treatment with a two-stage chem ical carcinogenesis protocol (40). In agreement with studies describing the ability of E2F1 to induce growth arrest, tumor suppressors p53 and p19ARF are necessary for deregulated E2F1 to inhibit tumor formation in this context (41). In line with deregulated E2F having c ontradictory biological effects in the regulation of cell cycle progression and tumo r development, deregulated E2F can also inhibit the induction of a poptosis under some contexts. As of yet this ability appears to be exclusive to instances of ra diation-induced apoptosis, and is thought to facilitate DNA

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19 repair (92,93). Transgenic expression of E2F1 to K5 expressing tissues suppresses epidermal apoptosis induced by UVB-irradi ation in a p53-indepe ndent manner (92,93). Furthermore, K5 E2F1 transgenic mice disp lay accelerated repair of UVB-induced DNA damage, indicating a role for E2F1 in promoting this type of DNA repair (92). While it is clear that deregulated E2F can promote grow th arrest, tumor suppression, or survival under some contexts, it appears as though the requ isite context is a no rmal cell absent of any losses of tumor suppressor function. Studi es utilizing E2F loss-of-function models better describe these effects a nd lend further support to the id ea that these contradictory biological effects are indeed important to normal physiology (94-99). However, the ability of deregulated E2F to inhibit cell growth, suppress tumor formation, and promote survival outside of the published contexts remains unclear. E2F target genes: connecting the biol ogy of deregulated E2F to mechanisms E2F family proteins have been implicated in controlling the expression of genes involved in functions as dive rse as DNA replication, the G1/S-phase transition, mitosis, DNA damage and repair, differentiation and development, and apoptosis (8,9,100). Some target genes have been thoroughly characteri zed by means of a comprehensive promoter analysis of E2F-mediated tran sactivation, or by inducing E2F activity in the presence of cyclohexamide, while others have been implicat ed in large-scale arra y-based analysis of E2F-induced transcripts or E2F-immunoprecipitated DNA. While a comprehensive review of all published E2F target genes involved in the many biological functions

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20 attributed to E2F is beyond the scope of this manuscript, target genes relevant to the subsequent experimental data are discussed in detail. Mitotic targets of E2F In addition to the well-characterized ro le of E2F-mediated transactivation of genes involved in the G1/S-phase transition and DNA rep lication, E2F has also been implicated in regulating the expression of cell cycle-associated genes with mitotic functions. Based on mircoarray analysis of transcripts, adenovirus-mediated overexpression of E2F1 or E2F2 in quiescent fi broblasts leads to th e induction of a large subset of genes with mitotic functions, such as kifC1, cdc2, cyclin B and cdc20 (101). Strikingly, a comparison of E2F1 and E2F2 i nduced transcripts to temporal regulation of whole genome transcripts during the cell cycle reveals targets of E2F1 and E2F2 to be physiologically induced primarily at either the G1/S transition or during G2suggesting a physiological role for E2F in the regulation of mitotic genes (101). While this study does not address whether the mitotic genes induced by E2F are di rect or indirect targets, chromatin immunoprecipitation (ChIP) of E2 F coupled to DNA microarray analysis reveals E2F present at the promoters of genes involved in chromatin assembly, condensation, and segregation, as well as the mitotic spindle checkpoint (102,103). A promoter based analysis of mitotic genes cell division control 2 (cdc2) and cyclin B1 reveals the presence of both positive and negative acting E2F elements, and that both E2F1 and E2F4 bind to the cdc2 and cyclin B1 promoters in vivo (104). Interestingly, E2F1 is only found at the cd c2 and cyclin B1 promoters during the G1/S-

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21 phase transition and S-phase, with E2F1 being completely disassociated by G2 (104). In addition to cdc2 and cyclin B1, the mitotic ch eckpoint protein mad2 is also a direct E2F1 target gene, which couples deregulated E2 F activity with the pr omotion of genomic instability (105). While a numb er of E2F targets with m itotic functions have been identified, only a handful have been characterized. Yet given the presence of E2F at the promoters of genes with mitotic functions, it would appear that E2 F-mediated regulation is at least in part a direct mechanism. This presents as so mewhat of a paradox, as E2F is thought to be no longer active when these genes are induced, and furthermore ChIP assays reveal E2F to be fully disassociated by G2 as well (104). While the precise mechanism by which E2F regulates the expression of genes with mitotic functions is yet to be determined, it is clear that E2F indeed plays a role that is in some instances direct. Apoptotic E2F targets and mechanisms In addition to promoting cell cycle progr ession, E2F1 is also a potent inducer of apoptosis, and as such many transcriptional targets of E2F1 have functional roles in various stages of this process. Whereas fe w of the mitotic targets of E2F are well characterized, much more is known about transcriptional targets and mechanisms of E2F1-induced apoptosis. Indeed, E2F1 is imp licated in the regulation of a multitude of genes with apoptotic functions; however th e following will concentrate on the bestcharacterized mechanisms. E2F1-induced apoptosis is generally categorized as occurring through either p53 family-dependent or p53 fa mily-independent pathways by means of

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E2F1 p19ARF Mdm2p53 Cellular Stress Apoptosis ATM p53-p p53 stabilization PUMA Apaf-1 p73 Bokp53 Family DependentMcl-1 hTERT Survival signals p53 Family independent Figure 4. Mechanisms of E2F1-induced apoptosis. E2F1 induces apoptosis through both p53 family-dependent and independent pa thways through both direct and indirect mechanisms. E2F1 indirectly stab ilizes p53 by transactivation of p19ARF or ATM. While p19ARF inhibits the activity of negative p53 regulator Mdm 2, ATM stabilizes p53 through phosphorylation. E2F1 can also induce the transcription of p53 homologue p73. E2F1 directly induces the transcrip tion of proapoptotic genes, su ch as Bok, Apaf-1 and PUMA, and can also directly repress the expression of prosurvival genes such as Mcl-1 and hTERT. 22

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23 three primary mechanisms: the indirect stabi lization of p53, the dire ct tranactivation of proapoptotic genes, and the direct repression of genes that promote ce ll survival (Fig. 4). While overexpression of E2F1 induces pr oapoptotic p53 (106), it does not do so directly and instead indirec tly stabilizes p53 protein through two separate mechanisms. In a healthy cell, p53 activity is kept in check pr imarily at the level of protein stability. In response to cellular stress, E2F1 can directly induce the expression of p19ARF (68,107), which in turn binds to and inhibits th e action of murine double minute 2 (Mdm2) (108,109). Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation, and as such the end result of E2F1-mediated transactivation of p19ARF is stabilization of p53 (110,111)which leads to p53-mediated transa ctivation of proapoptotic genes. In addition to regulation of p53 protein stability via the ubi quitin-proteasome pathway, p53 is also subject to stabilizi ng phosphorylations by stress-sensitive kinases. Stabilization of E2F1 in response to DNA damage results in E2F1-mediated direct tranactivation of Ataxia Telangiectasia-Mutated (ATM), whic h stabilizes p53 prot ein by phosphoylation at serine 15 (112,113). In addition to indirect stab ilization of p53, E2F1 can also directly induce transcription of p53 homologue p73 (114,115), whose activation can induce apoptosis in a manner similar to that of p53. In addition to p53-family dependent mechanisms of E2F1-induced apoptosis, E2F1 can also contribute to apoptosis th rough mechanisms independent of p53 family proteins. This can occur through two primary mechanisms: the direct transactivation of proapoptotic genes, and the dire ct or indirect repression of prosurvival genes. The use of microarray analysis of genes induced upon E2 F1 overexpression, as well as array-based analysis of E2F1-bound DNA by ChIP, has impli cated a multitude of potential apoptotic

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24 targets of E2F1. However, what is unclear fr om these analyses is the relevance of these genes in E2F1-induced apoptosis. For this r eason, the following will concentrate on those genes that are well characterized targets of E2 F1. E2F1 directly induces the expression of multiple Bcl-2 homology (BH)3-only proteins including PUMA, Noxa, Bim, Bik, and Hrk/DP5 (116,117), proapoptotic B-cell lympho ma/leukemia-2 (Bcl-2) family members that function in the intrinsically-mediated apoptotic pathway to promote the release of cytochrome c and other mitotic factors from the mitochondria. In addition to BH3-only Bcl-2 family targets, E2F1 also directly tr ansactivates the expre ssion of proapoptotic Bcl2 family member Bok (118), which also functions to compromise mitochondrial membrane integrity. Other notable direct ta rgets of E2F1 include Apoptosis activating factor-1 (Apaf-1) and Smac/DIABLO, as well as several caspases (119-121). Contrary to targets and mechanisms in which E2F1 induces the expression of genes that promote apoptosis, E2F1 can intri guingly also repress the expression of genes with prosurvival functions through both direct and indirect mechanisms. E2F1 directly represses transcription of antiapoptotic Bc l-2 family member Myeloid cell leukemia-1 (Mcl-1), which interestingly occurs in a pRb dependent manner, as deletion of the pRbbinding/transactivation domain does not abr ogate its ability (1 22). Similarly, E2F1 directly represses th e expression of human telomerase reverse transcriptase (hTERT), a gene involved in the maintenance of chromo some telomeres (123). In the death receptor mediated apoptotic pathway, TNF Receptor-A ssociated Factor 2 (TRAF2) inhibits apoptosis by stimulating antiapoptotic NF-kB. E2F1 can indirectly downregulate TRAF2 at the level protein though an as yet uncharacterized mechanism, providing yet another example of inhibition of survival genes me diated by E2F1 (124). Taken together, the

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25 preceding indicates that E2F1 plays a major ro le in regulating apoptosis through both p53 family-dependent and independent pathways functioning through multiple mechanisms. E2F targets and mechanisms involved in grow th arrest, tumor suppression, and survival E2F targets and mechanisms involved in grow th arrest, tumor suppression, and survival As previously discussed, under ce rtain contexts deregulated E2F can paradoxically promote survival, induce growth arrest, or contribute to tumor suppression (38-45). In many instances these biological e ffects are dependent on the presence of one or more tumor suppressors such as p19ARF, p53, p21, p16INK4A, or Rb (42-45). The mechanism by which E2F1 regulates p19ARF and ATM to ultimately control p53, as well as its direct ability to tr ansactivate p73, has been thor oughly discussed in a previous section, however in addition to these mechanis ms, other tumor suppressors are also direct targets of E2F. E2F can directly induc e the transcription of CKIs p21, p27, and p57, suggesting a negative-feedback mechanism limiting the activity of E2F (125-127). As exemplified by ATM, E2F can also influence the expression of multiple genes with roles in the DNA damage response and checkpoint control (3,4,8,100,128). This however leads to a rather complex web of functions, as many E2F targets involved in the DNA damage checkpoint and DNA repair also play roles in apoptosis and general DNA synthesis. In summary, under certain contexts deregulated E2F can induce th at transcription of genes that inhibit cell proliferation, promote surviv al, or suppress tumor formation, however the contexts determining preferential transcrip tion of these genes remains to be further explored.

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26 The RhoBTB2 (DBC2) putative tumor suppressor gene RhoBTB2, or Deleted in Breast Cancer 2 (DBC2) is a putative tumor suppressor gene located at 8p21 (129), a common spot for homozygous deletion in human malignancies arising from various tissues of origin (130-136). R hoBTB2 is the second member of a subclass within the Rho family of small GTPases proteins (RhoBTB1-3) and is highly divergent from other R ho family members. Orthologues of human RhoBTB genes are present in mammals, fish, flies and D. discoideum yet orthologues are absent from the genomes of yeast and worms (129,137). While only a handful of studies concentrating on RhoBTB2 have been publis hed, the following describes what is currently known. Structure RhoBTB2 is composed of an N-terminal RhoGTPase domain, two broad-complex bric-a-brac/poxvirus zinc finger (BTB/POZ ) domains, and a conserved C-terminal domain of unknown function. The RhoGTPase do main is highly homologous to that observed in other small GTPbinding proteins, and although it contains three putative GTP-binding motifs and a GTPase motif, studies indicate that it is incapable of GTP hydrolysis (138). In contrast to other members of the Rho family, RhoBTB2 contains a tandem of BTB/POZ domains, which are evolut ionarily conserved domains thought to be involved in protein-protein in teractions (139). BTB/POZ domains were first identified in Drosophila where such proteins act as transc riptional repressorsyet many BTB/POZ

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27 domain-containing proteins are encoded in the human genome (139). In humans, the BTB/POZ domains of RhoBTB2 as well as othe r proteins have been shown to interact with the Cullin 3 (Cul3) ubiquitin ligase complex, indicating a possible mechanism of regulation or action (140-144). Expression patterns During mouse embryogenesis, expression of RhoBTB2 mRNA is dependent on both tissue type as well as developmental st age (145). The highest levels of RhoBTB2 expression in the developing mouse embryo are in nervous system tissues, where elevated levels of expression contin ue until embryonic day E16.5when levels significantly decrease yet re main detectable throughout th e remainder of development (145). The developing gut and liver also display temporal increases in RhoBTB2 expression, yet surprisingly expression in the embryonic lung and mammary gland is very weak (145). This is intriguing, as dere gulation of RhoBTB2 in human malignancy is best documented in cancers of the lung and breast (146). Human multi-tissue arrays reveal RhoBTB2 expression to be weak in mo st tissues except neural tissues (129), while another study finds RhoBTB2 expression pres ent in noncancerous human breast, lung, brain, and placenta samples (146). Similarly, human fetal tissues show detectable RhoBTB2 expression in the lung, heart, a nd brain (129). Given the variability of expression patterns between studie s and the deficiencies in quan tification, it is difficult to make any concrete generalizations about R hoBTB2 expression patterns in developing or mature tissues.

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28 Deregulation in human malignancy Alteration of RhoBTB2 in human malignanc y has been described in the literature to occur by mean of deletion or loss of he terozygosity, downregulation, or point mutation (130-136,144,146). Indeed, RhoBTB2 was first characterized in humans by virtue of its deletion in primary breast cancer samples, wh ere it is reported to be heterozygously deleted in 3.5% of cas es (146). Deletions of RhoBTB2 have also been described in malignancies of the bladder, lung, ovary, and prostate (130-136), and ablation of RhoBTB2 expression through downr egulation is reported to o ccur in approximately 50% of breast and lung cancers (146). In additi on to deletion and downregulation, several point mutations have also been identified, w ith some of them effecting RhoBTB2 activity (144,146), although the biological si gnificance of this is yet to be determined. These studies would seem to suggest that RhoBTB2 might behave as a tumor suppressor, and this idea is indeed supported by limited biological studies. Biological functions, mechanisms, and regulation Given the prevalence of RhoBTB2 alte rations in human malignancy, one might suspect RhoBTB2 to behave biologically lik e a tumor suppressor, and indeed, the limited biological studies on RhoBTB2 support this hypothesis. Overexpression of RhoBTB2 in a breast cancer cell lin e with undetectable endogenous RhoBTB2 greatly inhibits proliferation, whereas overexpression in a cell line with endogenous RhoBTB2 has no effect on proliferation (146). Interestingly, overexpression of a BTB/POZ domain point-

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29 mutant RhoBTB2 construct derived from a human tumor (RhoBTB2-D299N) has no effect on proliferation, suggesting a role fo r the BTB/POZ domain in the mechanism of RhoBTB2-mediated cell cycle inhibition (146). In addition to inhibiting proliferation under certain contexts, RhoBTB2 has been link ed to the microtubule motor complex, as knockdown of RhoBTB2 in 293 cells abrogates vesicular stomatitis virus glycoprotein transport (138). Taken together, these studi es suggest that under some contexts, RhoBTB2 can function as a nega tive regulator of proliferat ion, and that RhoBTB2 has a functional role in transpor tation along the mictrotubule motor complex. However based on gain-of-function studies, it is not possible to classify RhoBTB2 as a tumor suppressor gene. A knockout mouse model is in order to fully examine the tumor suppressor capability of RhoBTB2. While the mechanism by which RhoBTB2 inhibits proliferation is not clear, downregulation of cyclin D1 has been propos ed. Overexpression of RhoBTB2 in a cell line deficient of endogenous RhoBTB2 expressi on leads to inhibiti on of cell cycle and downregulation of cyclin D1 protein, and overexpression of cyclin D1 upon RhoBTB2 overexpression ablates the ability of RhoBTB2 to inhibit pro liferation (147). It is clear that RhoBTB2 overexpression decreases cyc lin D1 protein, however the ability of enforced cyclin D1 overexpression to rescue ce lls from the inhibitory effect of RhoBTB2 does not demonstrate the necessity of cyclin D1 downregulation to mediate this process. It is likely that the enforced overexpression of many positive regulators of cell cycle would result in a similar effect. With this in mind, it is not clear if downregulation of cyclin D1 is a mechanism by which RhoBTB2 inhibits proliferation; studies utilizing cyclin D1 deficiencies would better address this issue. A microarray-based network

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30 analysis of transcripts influenced by R hoBTB2 deficiency and proficiency reveal RhoBTB2 to influence pathways responsible for cell cycle, apoptosis, cytoskeleton and membrane-trafficking, however the relevance of such conclusions is not clear (148). Taken together, while it is cl ear that RhoBTB2 influences the expression of various genes, the mechanisms by which RhoBTB2 i nhibits proliferation and influences the microtubule motor complex remain uncertain. Only one physiological means of RhoBTB 2 regulation has been reported in the literature, which involves degradation by th e proteasome. RhoBTB2 binds to the Cul3 ubiquiting ligase scaffold though it s first BTB/POZ domain and is also a substrate for the Cul3 ubiquitin ligase complex, which targ ets RhoBTB2 for degradation (144). A RhoBTB2 construct derived from a lung can cer cell line containing a point mutation (Y284D) in the first BTB/POZ domain abolis hes the ability of RhoBTB2 to bind Cul3, and thereby increases its expression due to de creased degradation. Th e authors present an attractive model in which the tumor suppre ssor function of RhoBTB2 is achieved via recruiting proteins to the Cu l3 ubiquitin ligase, thus targ eting them for proteasomal degradation; however this model is yet to be tested. Summary and rationale Given the prevalence of Rb-E2F pathway deregulation in human malignancy and the detrimental biological effects associated with unrestrained E2F activity, we sought to identify novel transcriptional targets of E2F1. In this ma nuscript, we identify RhoBTB2 as a novel transcriptio nal target of E2F1. We demonstr ate that overexpression of E2F1

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31 directly activates RhoBTB2 expression, a nd that knockdown of E2F1 decreases the expression of RhoBTB2, thus indicating that E2F1-mediated activation of RhoBTB2 is physiologically relevant and not simply an artifact of overexpression. Furthermore, we show that RhoBTB2 is upregulated during mitosis as well as during drug-induced apoptosis, and that this activation is pa rtially and primarily dependent on E2F1, respectively. Finally, we demonstrate that RhoBTB2 has active role s in E2F-mediated processes of cell cycle progression and apoptos is. Taken together, we describe RhoBTB2 as a novel transcriptio nal target of E2F1 with roles in cell cycle and apoptosis.

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32 Experimental Procedures Cell lines and cell culture The H1299 cell line was a gift from Dr Jiandong Chen (Moffitt Cancer Center, Tampa, FL) and cultured in DMEM supplemen ted with 2 mM L-glutamine, 5% fetal bovine serum (FBS) and 1% pe nicillin/streptomy cin (P/S). The MCF7 and MCF10A mammary fibrocystic cell lines were a gift from Dr. Richard Jove (C ity of Hope, Duarte, CA) and were cultured in DMEM-F12 supplemented with 2 mM L-glutamine, 10% FBS and 1% P/S. The T98G glioblastoma cell line was a gift from Dr. Joseph Nevins (Duke University, Durham, NC) and grown in DMEM supplemented with 2 mM L-glutamine, 10% FBS, and 1% P/S. The H1299-pBS/ U6 and H1299-shE2F1 cell lines were constructed and cultured as previously described (125,126,149). The H1299-ER-E2F1 cell line was constructed and culture d as previously described (125,126,149,150). Adenovirus The Ad-GFP and Ad-E2F1-GFP adenoviru s were kind gifts from Dr. Timothy Kowalik (University of Mass achusetts, Worchester, MA) ( 20,89). The Ad-E2F1(1-283)GFP adenovirus was constructed as previ ously described (42). The Ad-RhoBTB2-GFP

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33 adenovirus was constructed using a cDNA construct of RhoBTB2 with an N-terminal 3XFlag sequence and a C-terminal myc tag. The entire double-tagged sequence was used for virus construction with the AdEasy Ade noviral Vector System (Stratagene) using the pShuttle-IRES-hrGFP-1 vector following the manufacturers protocol. Titering was conducted using the AdEasy Vi ral Titer Kit (Stratagene). Real-time PCR Total cell RNA was harvested using the RNeasy Mini Kit (Qiagen) using the optional DNase treatment. Reverse Transcript ase (RT) reactions were random hexamerprimed using Applied Biosystems (Foster City, CA) High Capacity cDNA Archive Kit. Standard curves were constructed using serial dilutions of pooled sample RNA (50, 10, 2, 0.8, 0.4, and 0.08 ng) per reverse transcriptase reaction. One no reverse transcriptase control was included for the standard curve and for each sample. TaqMan Gene Expression Assays (Applied Biosystems) were used. The assay primer and probe sequences are prop rietary. TaqMan probe Hs01598093_g1 was used for RhoBTB2. Real-time quantitative PCR analyses were performed using the ABI PRISM 7900HT Sequence Detection System (A pplied Biosystems). All standards and samples were tested in triplicate wells. The no template control (H2O), no RT controls, no amplification control (Bluescript plasmid), and No RNA control were tested in duplicate wells. PCR was carried out with the TaqM an Universal PCR Master Mix (Applied Biosystems) using 2 l of cDNA and 1X pr imers and probe in a 20-l final reaction mixture. After a 2-min incubation at 50C, AmpliTaq Gold was activated by a 10-min

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34 incubation at 95C, followed by 40 PCR cycles consisting of 15s of denaturation at 95C and hybridization of probe and primers for 1 mi n at 60C. Data were analyzed using SDS software version 2.2.2 and exported into an Ex cel spreadsheet. The 18s data were used for normalizing gene values: ng gene/ng 18s per well. RhoBTB2 antibody production Affinity-purified rabbit polyclonal an tibody was generated toward a peptide corresponding to human RhoBTB2 am ino acids 673-687 (KEEDHYQRARKEREK) by Pacific Immunology (Ramona, CA). Sp ecifically, a 16-amino acid peptide (CKEEDHYQRARKEREK) was conjugated (via an artificial N-terminal cysteine residue) to Keyhole Limpet Hemocyanin a nd used to immunize rabbits. Serum was subjected to peptide column a ffinity purification prior to use in immunofluorescence. Antibody specificity was demonstrated usi ng a previously described RhoBTB2 siRNA (148). Plasmids, siRNA, and transfections RhoBTB2 siRNA was custom made (Amb ion) using a previously published RhoBTB2 siRNA (DBC2) sequence (148). siCONTROL non-targeting siRNA (Dharmacon) was used for all negative c ontrols. The siRNA was transfected using Lipofectamine 2000 (Invitrogen) following th e manufacturers protocol. The pBB14 membrane GFP plasmid was a kind gift from Dr. L.W. Enquist (P rinceton), constructed

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35 as previously described (151) and transf ected with Lipofectamine 2000 following the manufacturers protocol. Immunofluorescent microscopy Cells were grown on Lab-Tek II Cham ber Slides (Nunc), fixed with 4% paraformaldehyde, permeabilized with 0.5% Tr iton-X, then blocked with 2% BSA in PBS. The primary RhoBTB2 antibody was used at a 1:40 concentration, and the secondary antibody was Alexa Fluor 555 goat anti-rabbit Ig antibody (Molecular Probes) at a concentration of 1:2000. Cover slips were mounted using ProLong Gold antifade reagent with DAPI (Molecular Pr obes). Samples were viewed with a fully automated, upright Zeiss AxioImagerZ.1 microscope with a 40x or 63x /1.40NA oil immersion objective, and DAPI, FITC and R hodamine filter cubes. Equal exposure times were used for each sample. Images were produced using the AxioCam MRm CCD camera and Axiovision version 4.5 software suite. Flow cytometry Cells were detached from culture plates via trypsin, washed twice with PBS, and then fixed in 70% ethanol. The fixed cells were washed twice with PBS and treated with RNase A and propidium iodide (P I). PI staining was used to measure for cell cycle status using a Becton-Dickinson FACScan in strument and Cell Quest software.

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36 MTS assays For siRNA and adenovirus based experiments, the cells were first transfected or infected as described in the results section. After 24 hours of transfec tion or infection the cells were trypsinized, counted, and then pl ated in 96-well plates. The specific drug treatments were then administered and the MTS assays were conducted using a CellTiter 96 AQueous One Cell Proliferation Assay Kit (Promega) following the published protocol.

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37 Results E2F1 overexpression upregulates RhoBTB2 Using a microarray screen, we sought to identify novel targ ets of the E2F1 transcription factor. In this approach, we infected the H1299 cell line with adenovirus expressing either a green fluor escent protein control construc t (Ad-GFP) or a GFP-fused E2F1 construct (Ad-E2F1-GFP). RNA was harvested at 24 and 48 hours and processed for microarray analysis. Among the list of genes whose transcripts were found to be highly induced by E2F1 infection was RhoBTB2. To confirm the microarray results, we infected H1299s with either Ad-GFP, AdE2F1-GFP, or Ad-E2F1(1-283)-GFP, a deletion mutant of E2F1 that is lacking the transactivation domain (45). Using real-time polymerase chain reaction (PCR) to quantify RhoBTB2 mRNA expression, we found that Ad -E2F1-GFP infection does indeed induce RhoBTB2 transcript approximately 5 and 20fold compared to that of the Ad-GFP infection at the 24and 48-hour time points, respectively (Fig. 5A). Lack of RhoBTB2 activation by Ad-E2F1(1-283)-GFP infection co nfirms that upregulation of RhoBTB2 by E2F1 is dependent on E2F1s C-terminal transcription activa tion domain. Since all experiments conducted to this point employed the H1299 cell lin e, we wanted to ensure that RhoBTB2 activation by E2F1 was not cell line-dependent. To this end, we infected

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24 hrs. 48 hrs. Ad-GFP Ad-E2F1 Ad-E2F1 (1-283) H1299 24 hrs.48 hrs. T98G 0 1 2 3 4 5 6 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 24 hrs. 48 hrs. Ad-GFP Ad-E2F1 MCF7A BAd-GFP Ad-E2F1RhoBTB2/18S RhoBTB2/18S RhoBTB2/18SC24 hrs. 48 hrs. Ad-GFP Ad-E2F1 Ad-E2F1 (1-283) H1299 24 hrs.48 hrs. T98G 0 1 2 3 4 5 6 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 3 24 hrs. 48 hrs. Ad-GFP Ad-E2F1 MCF7A BAd-GFP Ad-E2F1RhoBTB2/18S RhoBTB2/18S RhoBTB2/18SC Figure 5. E2F1 overexpression upregulates RhoBTB2 mRNA. (A) H1299s were treated with either Ad-GFP, Ad-E2F1-G FP, or Ad-E2F1(1-283)-GFP adenovirus, harvested at 24 and 48 hours, with real -time PCR conducted to quantify RhoBTB2 mRNA relative to 18S. (B, C) MCF7s or T98G s were treated with either Ad-GFP or AdE2F1 with subsequent real-time PCR analys is for RhoBTB2 to 18S at 24and 48-hour time points. 38

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39 the T98G and MCF7 cell lines with either Ad-GFP or Ad-E2F1 and conducted real-time PCR as in the prior experiment. We observed upregulation of RhoBTB2 similar to that which was observed in H1299s, thus confir ming that RhoBTB2 upregulation by E2F1 overexpression is not cell lin e specific (Fig. 5B, C). In order to conduct protein-based studies of RhoBTB 2, we raised a polyclonal antibody against a 15 amino acid peptide sequen ce located within th e C-terminus. While the antibody was not able to recognize endogeno us RhoBTB2 protein in a denatured state by western blot, we were able to vi sualize endogenous RhoBTB2 protein via immunofluorescenct microscopy (IFM) (Fig. 6A). To confirm that the observed signal was not an artifact of non-specific bindi ng, we transiently knocked-down RhoBTB2 expression using small inhibitory RNA (siRNA) and assayed for expression using IFM. As shown in Figure 6B, knock-down of RhoB TB2 expression diminishes the observed RhoBTB2 signal, thus confirming th e specificity of the novel antibody. Having an antibody functional for RhoBTB 2 protein quantification, we sought to determine if the observed upregulation of RhoBTB2 mRNA by E2F1 overexpression resulted in a corresponding increase of RhoB TB2 at the protein level. To this end, an HA-tagged version of E2F1 (HA-E2F1), as well as a GFP-expression vector, were cotransfected into H1299s. After 24 hours the cells were stained for RhoBTB2, and GFP positive and negative cells were used to sel ect for transfected a nd non-transfected cells, respectively. We found that cells positive for GFP (transfected) expressed a substantially higher level of RhoBTB2 protei n as compared to adjacent GFP-negative cells (Fig. 7), thus confirming that E2F1 overexpression re sults in increased expression of RhoBTB2

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RhoBTB2 DAPI Merge H1299 siControl siRhoBTB2 RhoBTB2 DAPI Merge A B Figure 6. Novel RhoBTB2 antibody is functional in immunofluorescent microscopy and is specific for RhoBTB2. (A) Immunofluorescent micr oscopy (IFM) of H1299s at 40x for RhoBTB2 with a rabbit polyclona l antibody described in experimental proceduresDAPI: blue; RhoBTB2: red. (B) IF M as in 6A of H1299s transfected with either negative control siRNA (top) or siRNA to RhoBTB2 (bottom) after 48 hours. 40

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41 protein. Taken together, these results demonstrate that RhoB TB2 is upregulated at both the mRNA and protein levels by E2F1 overexpression. Upregulation of RhoBTB2 by E2F1 is direct and not depende nt on artificial overexpression We considered the possibility that RhoBTB 2 might be an indirect target of E2F1; to address the issue of direct versus indirect activation, we utilized a well characterized H1299 cell line with an estrogen receptor-fused version of E2F1 stably integrated (H1299 ER-E2F1) (125,126,149,150). The result is an over expressed version of E2F1 that is transcriptionally inactive due to estrogen receptor-mediated cytoplasmic localization. Using this system, E2F1 activity can be ra pidly induced through nuclear localization by addition of the estrogen receptor li gand 4-hydroxytamoxifen (4-OHT), while simultaneously blocking new protein synthesis by means of cyclohexamide (CHX). Any transcripts found to be induced by 4-OHT in the presence of CHX can be considered direct E2F1 targets. As shown in figure 8, RhoBTB2 mRNA e xpression is relatively low in the untreated H1299 ER-E2F1 cell line, as well as after 8 and 24 hours of treatment of CHX alone. As expected, upregulati on of RhoBTB2 is readily obs erved at 8 and 24 hours after promoting E2F1 nuclear localization through treatment with 4-OHT. This activation of RhoBTB2 transcription by 4-OHT is not abr ogated upon co-administration of CHX, thus confirming that RhoBTB2 is a direct transcriptional target of E2F1.

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RhoBTB2 DAPI Merge GFP Transfection: HA-E2F1 + GFP RhoBTB2 DAPI Merge GFP Transfection: HA-E2F1 + GFP Figure 7. E2F1 overexpression upregulates RhoBTB2 protein. IFM at 63x of two different fields of H1299s 48 hours after be ing transiently cotran sfected with pcDNA3HA-E2F1 and pBB14, a membrane GFP plasmi dDAPI: blue; GFP (transfected cells): green; RhoBTB2: red. H1299 ER-E2F1 0 1 2 3 4No Treatment C HX O HT C H X + O HT CHX OH T CHX + O HT 8 hrs 24 hrs RhoBTB2/18S H1299 ER-E2F1 0 1 2 3 4No Treatment C HX O HT C H X + O HT CHX OH T CHX + O HT 8 hrs 24 hrs RhoBTB2/18S Figure 8. E2F1-mediated upregulation of RhoBTB2 is direct. The H1299-ER-E2F1 cell line was treated with either CHX, 4-OHT or both. Cells were harvested for real-time PCR analysis at 8a nd 24-hour time points. 42

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43 Having shown the capability of artifici ally overexpressed E2F1 to directly activate RhoBTB2, we next sought to determ ine if E2F1 plays a role in RhoBTB2 regulation under physiological conditions. To this end, we employed H1299 cell lines with a stably integrated short-hairpin inhibitory R NA corresponding to E2F1 (H1299shE2F1) or an empty vector contro l (H1299-pBS/U6) (125,126,149). We observed significant knockdown of E2F1 in the H 1299-shE2F1 in comparison to the H1299pBS/U6 as previously reported (Fig. 9A) (125,126,149). We stained the cells for RhoBTB2 and compared expression levels be tween the two lines by means of IFM. The H1299-pBS/U6 control cell line with unaltere d E2F1 expressed RhoBTB2 at levels comparable to that of the parental H1299 lin e (Fig. 9B). In contrast, the H1299-shE2F1 cell line displayed greatly diminished expres sion of RhoBTB2 when compared to that observed in the H1299-pBS/U6 cell line (F ig 9B). Given that knock-down of E2F1 diminishes RhoBTB2 expression, we conclude that E2F1 is indeed a physiological regulator of RhoBTB2. RhoBTB2 is upregulated during mitosis, which is partially dependent on E2F1 One of the main functions of the grow th promoting E2Fs is to activate the transcription of genes critical for cell cycle progression (8,9 ). Having identified RhoBTB2 as an E2F1 target gene, we pos tulated that RhoBTB2 expression may be regulated through this process. To examin e RhoBTB2 expression through the cell cycle, we stained an asynchronously growi ng population of H1299s for RhoBTB2 and examined the population for cells in interphase as well as various stages of mitosis via

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RhoBTB2 DAPI Merge H1299shE2F1 H1299pBS/U6 WB: E2F1H 1 2 9 9 p B S / U 6 H 1 2 9 9 s h E 2 F 1 RhoBTB2 DAPI Merge H1299shE2F1 H1299pBS/U6 WB: E2F1H 1 2 9 9 p B S / U 6 H 1 2 9 9 s h E 2 F 1 A B Figure 9. RhoBTB2 is a physiological target of E2F1. (A) A western blot for E2F1 in the H1299-pBS/U6 and H1299-shE2F1 cell lin es demonstrating efficient knockdown of E2F1. (B) IFM at 63x using the RhoB TB2 polyclonal antibody conducted on asynchronously growing H1299-pBS/U6 and H1299-shE2F1 cell linesDAPI: blue; RhoBTB2: red 44

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45 IFM. As shown in figure 10, H1299s in interphase express a relati vely low level of RhoBTB2; however, upon the initiation of prophase R hoBTB2 levels increase dramatically. RhoBTB2 expression remain s highly elevated through metaphase and anaphase, and does not begin to de crease until telophase/cytokinesis. A vast majority of cancers exhibit aberrant regulation of the RB-E2F pathway, with the end result being unrestrained E2F mol ecules. We considered the possibility that the observed mitotic upregul ation of RhoBTB2 may be an artifact of the highly transformed H1299 phenotype. To address this issue, we conducted identical experiments in MCF10As, a non-tumorigenic mammary fibroc ystic cell line. In these experiments we observed mitotic upregulation of RhoBTB2 that parallels that observed in H1299s (Fig. 10), confirming that upregulation of RhoBTB2 during mitosis is not due to the highly transformed nature of H1299s. We next wanted to determine if the observed mitotic upregulation of RhoBTB2 was dependent upon E2F1. We utilized the aforementioned E2F1 proficient and knockdown cell lines H1299-pBS/U6 and H 1299-shE2F1 to compare cell cycle regulation of RhoBTB2 in cel ls with two different le vels of E2F1 expression. Asynchronously growing populations of the two cell lines were stained for RhoBTB2 and examined for cells in interphase and various stages of mitosi s as previously described. As expected, mitotic upregulation of RhoBTB2 was readily observed in the H1299-pBS/U6 cell line, and was comparable to that seen in the parental H1299s (Fig. 11, top panel). During interphase, the H1299-shE2F1 cell line has lower basal expression of RhoBTB2, as previously observed. However, we not ed an impaired mitotic upregulation of RhoBTB2 in the H1299-shE2F1 cell line (Fi g. 11, bottom panel). While there is an

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InterphaseProphaseMetaphaseAnaphaseTelophase MCF10A H1299 DAPI RhoBTB2 Figure 10. RhoBTB2 is upregulated during mitosis. IFM at 63x using the RhoBTB2 polyclonal antibody of representative H1299s (t op) and MCF10A (bottom) cells in either interphase, prophase, metaphase, anapha se, or telophase/cytokinesis within aynchronously growing populations DAPI: blue; RhoBTB2: red. InterphaseProphaseMetaphaseAnaphaseTelophase H1299shE2F1 H1299pBS/U6 DAPI RhoBTB2 Figure 11. Mitotic upregulation of RhoBTB 2 is partially dependent on E2F1. IFM at 63x using the RhoBTB2 polycl onal antibody of representative H1299-pBS/U6 (top) and H1299-shE2F1 (bottom) cells in either in terphase, prophase, meta phase, anaphase, or telophase/cytokinesis within aynchro nously growing populationsDAPI: blue; RhoBTB2: red. 46

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47 evident upregulation of RhoB TB2 during prophase, it is significantly impaired when compared to that observed with the E2F1 proficient H1299-pBS/U6 cell line. This trend of diminished mitotic upregulation of R hoBTB2 continues to be observable throughout all of the mitotic phases examined (Fig. 11). We postulate that residual regulation ofRhoBTB2 may be mediated by additional E2 F family members. Taken together, this demonstrates that RhoBTB2 is indeed upregulated during mitosis, which is partially dependent on the presence of E2F1. Overexpression of RhoBTB2 increases the S-phase fraction and slows proliferation Given the observation that RhoBTB2 is upregulated during M-phase of the cell cycle, we sought to determine if artificial manipulation of RhoBTB2 levels would have a functional and observable effect on cell cycle status or proliferation. To this end, we constructed an adenovirus expressing either GFP (Ad-GFP) or RhoBTB2 fused to an internal ribosome entry s ite (IRES) GFP construct (AdRhoBTB2-GFP). Asynchronously growing H1299s were then infected with equal amounts of either Ad-GFP or AdRhoBTB2-GFP and harvested at 48 hours for flow cytometric analysis of cell cycle status via propidium iodide (PI) staining. As shown in figure 12A, overexpression of RhoBTB2 alters the cell cycle status of H1299s by increasing the fr action of cells in S-phase. Having noted that overexpression of RhoBTB 2 increased the S-phase fraction; we wanted to know how this singl e snap shot of cell cycle stat us manifested in a functional effect on cell proliferation. To test this, we infected asynchronously growing H1299s with either Ad-GFP or Ad-RhoBTB2-G FP adenovirus and conducted MTS-based

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A B 01000200030004000 Propidium Iodide 0 100 200 300 400 500 # Cells 01000200030004000 Propidium Iodide 0 200 400 600 800 # Cells G0/G1 : 48.38 +/-0.65 S : 35.09 +/-1.80 G2/M : 16.36 +/-2.12Ad-GFP Ad-RhoBTB2-GFP 0 20 40 60 80 24 hr 48 hr 72 hr Ad-GFP Ad-RhoBTB2-GFP% cell increaseG0/G1 : 36.69 +/-1.27 S : 54.38 +/-0.75 G2/M : 8.93 +/-0.56 Figure 12. Overexpression of RhoBTB2 increa ses the S-phase fraction and slows proliferation. (A) The H1299 cell line was infected in triplicate with equal amounts of either the Ad-GFP or Ad-RhoBTB2-GFP adenovirus and harvested 48 hours postinfection for flow cytometry. Propidium Iodide was used to analyze cell cycle status. (B) H1299s were infected in tr iplicate with equal amounts of either the Ad-GFP or AdRhoBTB2-GFP adenovirus, detached at 24 post-infection, counted, a nd transferred to 96 well plates where an MTS assay was performe d to analyze cell pro liferation after 24, 48 and 72 hours. 48

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49 proliferation assays. As shown in figure 12B cells infected with RhoBTB2 adenovirus exhibited impaired cell proliferation over multip le passages as compared to those infected with the control GFP virus. Since we observed an increase in the S-phase fraction as well as slowed cell progre ssion upon overexpression of RhoBTB 2, we wanted to determine ifsiRNA-mediated knockdown of RhoBTB2 wo uld alternatively decrease the S-phase fraction or increase the rate of proliferation. We found that depletion of RhoBTB2 did not alter the cell cycle status or the rate of pro liferation, consistent with the idea of RhoBTB2 as being a negative regulator. From these observations, we conclude that the observed increase in the S-phase fraction upon overe xpression of RhoBTB2 is potentially caused by a transient S-phase arre st or lengthened S-phase. RhoBTB2 is upregulated during drug-induced apoptosis, which is primarily dependent on E2F1 E2F1 is unique among the E2F family memb ers in that it not only has the ability to transactivate genes critical for cell cycle progression, but is also a potent inducer of apoptosis through activati ng the transcription of proapoptotic genes (f or review, see ref. (24)). Given this fact, we investigated whether RhoBTB2 expression was effected by drug-induced apoptosis. To determine whet her RhoBTB2 is regulated by apoptotic insults, we treated H1299s with either cisplatinum, flavopi ridol or etoposide, chemotherapeutic agents where E2F1 is know n to be a critical mediator, and conducted IFM to determine whether these cytotoxic in sults had any effect on RhoBTB2 expression. As shown in figure 13A, we observed that administration of all of the chemotherapeutic

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DAPI RhoBTB2 Merge Flavopiridol Cisplatinum No Drug Etoposide DAPI RhoBTB2 Figure 13. RhoBTB2 is upregulated during drug-induced apoptosis. IFM at 63x using the RhoBTB2 polyclonal antibody of representative cells from H1299s after 24 hours of either no treatment, 20 uM cisp latinum, 200 nM flavopiridol, or 20 uM etoposideDAPI: blue ; RhoBTB2: red. 50

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51 agents tested resulted in increased R hoBTB2 protein expression, with flavopiridol exhibiting the weakest. While we observed upregulation of RhoBTB 2 during cytotoxic insult, we wanted to determine if E2F1 was responsible for th is upregulation. To examine this issue, we utilized the previously described E2F1 proficient and knockdown cell lines H1299pBS/U6 and H1299-shE2F1 and conducted IFM on cells treated with the aforementioned apoptotic stimuli. As previously observed, RhoBTB2 expression was diminished in the untreated H1299-shE2F1 cell line compared to the control H1299-pBS/U6 cell line (Fig. 14A). Upon the induction of apoptosis, the control H1299-pBS/U6 cell line behaved similar to that of the parental H1299s, with upregulation of RhoBTB2 being clearly evident after 24 hours (Fig. 14, top). In st ark contrast, we observed very little upregulation of RhoBTB2 in the H1299-shE2 F1 cell line (Fig. 14A, bottom). Figure 14C displays E2F1 protein levels at 24 hours post treatment, demonstrating that E2F1 upregulation does not occur in the H1299-shE2 F1 cell line even in the presence of cytotoxic insult. It should be noted that in the presence of flavopiridol, we observe upregulation of E2F1 to be highest s hortly after treatment (around 6 hours) and diminished by 24 hours, which explains the seemingly diminished E2F1 expression as compared to the no treatment control. Taken together, these results demonstrate that RhoBTB2 is upregulated during drug-induced apoptosis, and that this upregulation is primarily dependent on the presence of E2F1.

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H1299shE2F1 H1299pBS/U6 No TreatmentCisplatinumFlavopiridolEtoposide N o T r e a t m e n t C i s p l a t i n u m F l a v o p i r i d o l E t o p o s i d e No T r e a t m e n t C i s p l a t i n u m F l a v o p i r i d o l E t o p o s i d eH1299-shE2F1 H1299-pBS/U6A BDAPI 52 RhoBTB2 H1299shE2F1 H1299pBS/U6 No TreatmentCisplatinumFlavopiridolEtoposide N o T r e a t m e n t C i s p l a t i n u m F l a v o p i r i d o l E t o p o s i d e No T r e a t m e n t C i s p l a t i n u m F l a v o p i r i d o l E t o p o s i d eH1299-shE2F1 H1299-pBS/U6A BDAPI RhoBTB2 Figure 14. Upregulation of RhoBTB2 during drug-induced apoptosis is primarily dependent on E2F1. (A) IFM at 63x using the RhoBTB2 polyclonal antibody of representative H1299-pBS/U6 (top) or H1299-shE2F1 (botto m) cells after 24 hours of either no treatment, 20 uM cisplatinum, 200 nM flavopiridol, or 20 uM etoposide DAPI: blue; RhoBTB2: red.

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53 Knockdown of RhoBTB2 expression by siRN A impairs the induction of drug-induced apoptosis Previous experiments demonstrated th at RhoBTB2 is upregulated during druginduced apoptosis in an E2F1-dependent manne r; we therefore wanted to explore whether disruption of RhoBTB2 activity would ha ve a functional effect on drug-induced apoptosis. To address this issue, we transi ently depleted RhoBTB2 in H1299s via siRNAmediated knockdown of RhoBTB2, induced apoptosis using the drug treatments previously employed, and conducted MTS assays to measure cell viability over the span of three days. While loss of viability o ccurred in both the si Control and siRhoBTB2 transfected cell lines upon cytotoxic drug treatmen t, this loss of viability was abrogated in cells lacking RhoBTB2 (Fig. 15). We observed similar results in all drug treatments used, implying that RhoBTB2 may play a more ubiqui tous role in apoptosis. Since we observed abrogated induction of apopt osis in upon siRNA-mediat ed knockdown of RhoBTB2, we wanted to determine if overexpression of RhoBTB2 would alternatively hasten the induction of apoptosis. We found that ade novirus-mediated overe xpression of RhoBTB2 did not hasten the induction of apoptosis. We interpret these data as for the first time demonstrating that RhoBTB2 plays a direct a nd important role in the implementation of apoptosis.

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A B C Cisplatinum 50 60 70 80 90 100 0 hr24 hr48 hr72 hr siControl siRhoBTB2 Flavopiridol 50 60 70 80 90 100 0 hr24 hr48 hr72 hr siControl siRhoBTB2 Etoposide 50 60 70 80 90 100 0 hr24 hr48 hr 72 hr siControl siRhoBTB2% viable % viable % viable Figure 15. Knockdown of RhoBTB2 via si RNA impairs the induction of druginduced apoptosis. H1299s were transiently transfected with either a negative control siRNA, or siRNA against RhoBTB2, det ached at 24 post-transfection, counted, and transferred to 96 well plates where an MTS a ssay was performed to analyze cell viability after 24, 48 and 72 hours of treatment with either 20 uM cisplatinum (A), 200 nM flavopiridol (B), or 20 uM etoposide (C). 54

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55 Discussion E2F is perhaps best known for its ability to promote the tran scription of genes involved in the G1/S-phase transition; however an increasing amount of evidence implicates a role for E2F in the regul ation of genes with mitotic functions. Overexpression of E2F1 or E2F2 induces a s ubset of genes with mitotic functions, and E2F1 can be found at the promoters of genes with mitotic functions (101-105). Furthermore, targets of E2F1 and E2F2 tend to be physiologically regulated temporally at two distinct cell cycle stag es: G1/S and G2, implicating a role for E2F-mediated transcription long after E2F is thought to be inactive (101). While a number of mitotic E2F targets have been identified, few have been characterized. In this work, we demonstrate that RhoBTB2 is a direct target of E2F1 that is physiologically upregulated du ring mitosis. We further show that mitotic upregulation of RhoBTB2 is partially dependent of E2 F1, as knockdown of E2F1 expression via shRNA abrogates mitotic upregulation of R hoBTB2. It is possible that the remaining mitotic upregulation of RhoBTB2 in the abse nce of E2F1 is dependent on E2F2 or E2F3a; however we have not pursued this hypothesis. In addition to being a mitotic target of E2F1, we also find that RhoBTB2 is an apoptotic target of E2F1 as well. RhoB TB2 is upregulated upon treatment with chemotherapeutic drugs, which is primarily independent on E2F1 as knockdown of E2F1

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56 with shRNA abrogates this effect as well. We see a greater dependence on E2F1 for apoptosis-induced upregulation as opposed to mitotic upregul ation, and this may be due to an inability of E2F2 or E2F3a to comp ensate, as E2F1 is the primary inducer of apoptosis among the activating E2Fs. In order to further explore the signi ficance of E2F-mediated regulation of RhoBTB2, we examined a functional role for RhoBTB2 in either of these processes. Overexpression of RhoBTB2 increases the frac tion of cells in S-phase and significantly impairs cell proliferation, which we interpre t as possibly being a transient S-phase block, as we only see a partial block in cell proliferation. In the case of apoptosis, we find that depletion of RhoBTB2 by siRNA slows the induction of drug-induced apoptosis. While deciphering mechanisms by which RhoBTB2 acts in cell cycle inhibition and the induction of apoptosis was beyond the sc ope of this study, published reports on RhoBTB2 have led to some intriguing hypotheses. In agreement with our observations RhoBTB2 was show n to inhibit cell proliferation in a breast cancer cell line deficient for RhoBTB2 (146). Futher studies asserted that RhoBTB2-mediated downregulat ion of cyclin D1 was obligatory for this effect (147). Another study u tilizing pathway-based analysis of gene expression patterns found RhoBTB2 to effect the expression of ge nes associated with cell cycle, apoptosis, cytoskeleton and membrane-trafficking pathways (148). But perhaps the most intriguing study of found that RhoBTB2 direct bound a nd was a substrate of the Cul3 ubiquitin ligase (144). The authors proposed a hypothesis in which RhoBTB2 served as a scaffold that recruited proteins to the Cul3 complex to be targeted for degradation. This seems

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57 quite rational, as other BTB/POZ domain-c ontaining proteins have similar functions (140-144). Given the previously mentioned studie s coupled with our own observations, we believe that the functional significance of E2F1-mediated upregulation of RhoBTB2 could be directly related to the ability of RhoBTB2 to r ecruit proteins to the Cul3 complex to be targeted for degradation. We propose a model in which the physiological role of RhoBTB2 in mitosis and apoptosis is to recruit proteins to the Cul3 complex to be targeted for degradation, and that the ce ll cycle inhibition obser ved during overexpression may be a non-physiological response from RhoB TB2 targeting proteins to Cul3 in phases of the cell cycle where RhoBTB 2 would not normally be present (Fig. 16). While cyclin D1 would seem like an attractive candidate to mediate this effect, one would not expect to see an arrest occurring in S-phase or G2/M upon loss of cyclin D1. Additionally, cyclin D2 or D3 might be expected to compensate. While the mechanisms behind the biological functions of RhoBTB2 are yet to be determined, it is cl ear that RhoBTB2 is indeed a physiologically releva nt direct target of E2F1.

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RhoBTB2 RhoBTB2 Cul3 Cul3 RING RING NEDD8 Catalytic Core Substrate Specificity Ubiquitin 26S Proteasomal Degradation Protein X Protein X Protein X Protein X Cell Cycle and Apoptosis Regulatory Protein(s) Figure 16. A proposed mechanisti c model for RhoBTB2 activity. In this model, we propose that RhoBTB2 exerts its cell cycle and apoptotic biolog ical effects by facilitating uibiquitination and subsequent degradati on of cell cycle and apoptosis regulatory proteins. We propose that RhoBTB2 acts as a substrate-specific adaptor for the Cul3 ubiquitin ligase. 58

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59 PART II IDENTIFICATION AND CHARACTERIZ ATION OF TWO NOVEL MCL-1 PROMOTER POLYMORPHISMS

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60 Abstract A publication from Moshynska et al. identi fied two novel sequence variants of the MCL-1 promoter within lymphocytes from chronic lymphocytic leukemia patients (CLL), but not within noncancerous tissue from the same individuals or in lymphocytes from 18 healthy control subjects (1). This resu lt suggested that the va riantsinsertions of 6 or 18 nucleotides at position relative to the transcription start sitewere CLLrelated somatic oncogenic mutations. Moshynska et al. also determined that the 6and 18-nucleotide insertions were associated el evated Mcl-1 expression, and proposed that the variant promoters could be used as a prognostic marker. We independently identified and cloned the three observed sequence variants from cancer cell lines hereby referred to as the Mcl-1 +0, +6 or +18 promoters. In contrast to Moshynska et al., we find the variant promoters to be identically present in bot h cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line en coded. We also find the three variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that obs erved in cancerous cell lines. Furthermore, activity analysis of the thr ee variant promoters reveals th e Mcl-1 +6 and +18 promoters to be less active than the Mcl-1 +0 prom oter, both during normal cellular homeostasis and under conditions that actively induce Mcl1 transcription. Given our results, we

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61 conclude that the Mcl-1 +6 and +18 promoters are likely benign polymorphisms and do no represent a reliable prognostic marker.

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62 Introduction Mcl-1 and the Bcl-2 family of proteins Mcl-1 is an antiapoptotic member of th e Bcl-2 family of proteins. The Bcl-2 family is a group of proteins involved in th e intrinsic stress-media ted apoptotic pathway whose primary role is to regul ate the release of cytochrome c and other apoptotic factors from the mitochondria and possibly the endoplasmic reticulum (ER) (152,153). Upon release from the mitochondria, cytochrome c forms a complex with Apaf-1 which cleaves and activates effector caspases, thus initia ting apoptosis (154,155). The Bcl-2 family is divided into three subfamilies that play distin ct roles in both promoting and inhibiting the integrity of the mitochondrial membrane and ultimatelythe release of cytochrome c and the initiation of apoptotsis. These subfam ilies consist of the proapoptotic BH3-only subfamily, the antiapoptotic Bcl-2 subfamily, and the proapoptotic Bax subfamily. The roles of each family member in the intrinsi c apoptotic pathway are illustrated in figure 17.

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Cellular Stress BH3-only Bcl-2 Bax Cytochromec Release Figure 17. The Bcl-2 family and the intr insic stress-induced apoptotic pathway. Cellular stress activat es proapoptotic BH3-only Bcl2 subfamily members through various mechanisms. BH3-only subfamily me mbers block the ability of antiapoptotic Bcl-2 subfamily members to restrain th e activity of proapoptotic Bax subfamily members. This leads to oligomerization of Bax subfamily members in the mitochondrial membrane, which promotes the release of cyto chrome c and other apoptotic factors, thus initiating apoptosis. 63

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64 The BH3-only subfamily The BH3-only subfamily consists of multiple family members that are the first responders to cellular stress w ithin the intrinsic apoptotic pathway (Fig. 13). Depending on the nature of the cellular stress, individua l or multiple BH3-only proteins may become activated. Activation can occu r through transcrip tional regulation, pos t-translational modification, or both, and is largely fa mily member-dependent. These subfamily members are proapoptotic, and promote the release of apoptotic factors from the mitochondria. While there is significant di sagreement as to the exact mechanism by which Bcl-2 family members interact to disrupt mitochondrial membrane integrity, it can be generally stated that the primary role of the BH3-only proteins is to antagonize the inhibitory action of antiapoptotic Bcl-2 fa mily members through direct binding. Indeed, the BH3-only family is named such due to the presence of a single BH3 domain which, depending on the subfamily member, binds to the receptor domain of one or more antiapoptotic Bcl-2 subfamily proteins (152,153). The Bcl-2 subfamily The second subfamily within the Bcl-2 fa mily is the Bcl-2 subfamily, and their primary role is to promot e cell survival. Like the BH 3-only subfamily, the Bcl-2 subfamily consists of multiple members; however the most notable and likely most relevant members are Bcl-2, Mcl-1 and Bcl-xL. Bcl-2 subfamily proteins act downstream of BH3-only proteins and, in the absence of activated BH3-only proteins, maintain

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65 mitochondrial membrane integrity at least in part by antagonizing the activity of Bax subfamily members. Structurally, Bcl-2 s ubfamily members contain a transmembrane domain (TM), and BH1-4 domains. The TM domain is thought to function as an anchor for integration into the memb ranes of the mitochondria and endoplasmic reticulum, and BH1, BH2 and BH3 domains collectively form a receptor domain specific for the BH3 motif. In the absence of cellular stress, Bcl-2 subfamily proteins are thought to restrain the proapoptotic activity of Bax subfamily proteins at least in part through direct binding of the Bcl-2 receptor domain to the BH3 doma in of Bax subfamily members. However in the presence of cellular stre ss and activated BH3-only protei ns, the BH3 domain of BH3only proteins is thought to di rectly bind to the receptor domain of Bcl-2 subfamily members and prevent Bcl-2 subfamily member s from restraining the activity of Bax or Bak (152,153). The Bax subfamily The final subfamily within the Bcl-2 fa mily is the proapoptotic Bax subfamily, which consists of only two members: Ba x and Bak. Bax and Bak are thought to be functionally redundant, as inactiv ation of either family member alone has little effect on apoptotsis, while inactivation of both significantly inhibits a poptosis (156). Structurally, Bax and Bak contain a TM domain and BH1-3 domains. Cellular stress induces Bax and Bak to make a conformational change and form homo-oligomers within the mitochondrial membrane. Oligomerization of Bax and Bak in the mitochondrial membrane disrupts membrane integrity and prom otes the release of a poptotic factors. The

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66 mechanism by which oligomerized Bax and Bak compromises mitochondrial membrane integrity has not been fully elucidated, although several models have been proposed (152,153). Mcl-1 is an inhibitor of apoptosis As illustrated in the previously describe d model, the physiological role of Mcl-1 and other antiapoptoti c subfamily members is to pr omote survival through the maintenance of mitochondrial membrane integrity (Fig. 13). This function is well documented in both cell culture-based and in vivo experiments utilizing both gain-offunction and loss-of-function techniques. While the experiments that lend support for this model of activity are best described for Bcl2, studies focusing on Mcl-1 will be the topic of discussion in the following paragraphs. In cell culture-based assays, Mcl-1 ov erexpression inhibi ts the induction of apoptosis in multiple models, and is exemplif ied by the ability of overexpressed Mcl-1 to inhibit apoptosis induced by st aurosporin or transient c-My c overexpression in Chinese hamster ovary cells (157,158). Additionall y, in murine myeloid progenitor cells, overexpression of Mcl-1 delays apoptosis induc ed by cytotoxic agents or growth factor withdrawal (159). In agreement with in vitro studies, transgenic expression of Mcl-1 in hematolymphoid tissues results in increased viability in various cells of lymphoid and myeloid originoccuring at both mature and immature stages of development (160,161). Transgenic expression of Mcl1 also promotes the developm ent of certain hyperplasias and malignancies, which may be the result of an inhibition of apoptosis (160,162).

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67 Studies dealing with the e ffects of deregulated Mcl-1 expression on aberrant tissue proliferation are addressed in more detail in the section regard ing Mcl-1 and oncogenic transformation. Similar to overexpression studies, depletion of Mcl-1 by means of antisense RNA or siRNA in cell culture assays can either promote spontaneous apoptosis or sensitize to apoptosis. This effect is well documented and holds true in multiple in vitro model systems. Indeed, several studies demonstrate the feasibility of utilizing siRNA against Mcl-1 as a therapeutic intervention in ma lignancy (163-166). Since disruption of both Mcl-1 alleles in mice results in peri-implant ation embryonic lethal ity (167), various targeted disruptions have been developed to examine the role of disrupted Mcl-1 expression in vivo Targeted deletion of Mcl-1 in the T or B cell lineages results in a significant reduction of B and T lymphocytes, and when deleted in the same lineage during lymphocyte development, increased apoptosis and developmental arrest is observed (168). Furthermore, deletion of Mcl-1 in mature lymphocytes leads to a loss of viability (168). Induced deletion of Mcl-1 in mature mice leads to depletion of the bone marrow due to decreased cell survival (169), and targeted deletion to macrophages and neutrophils results in decreased neutrophil surv ival as manifested by an increased rate of apoptosis in granulocytic compartments ( 170). Collectively, these experiments describe the crucial role that Mcl-1 play s in regulating apoptosis both in vitro and in vivo

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68 Mcl-1 and oncogenic transformation One of the defining features of malignant transformation is an ability to evade apoptotic signals. Given Mcl-1s potent ability to promote survival, it is not surprising that Mcl-1 can contribute to oncogenic tran sformation under certain contexts. Much of the support for this stems from experiment s utilizing mice transgenic for Mcl-1. Longterm expression of transgenic Mcl-1 target ed to hematolyphoid tissues results in the development of B-cell lymphoma (160), and explantation and culture of myeloid cells derived from Mcl-1 transgenic mice in th e presence of interleukin (IL)-3 can induce immortalization (161). Additionally, transgenic expression of murine Mcl-1 leads to islet cell hyperplasia (162). It is unlikely that Mcl-1 action al one is sufficient for oncogenic transformation and likely cooperates with one or many oncogenic mutations to promote characteristics of a malignant phenotype. It is clear however, that under the proper conditions, enforced Mcl-1 expression can c ontribute to the development of an oncogenic phenotype. Mechanisms regulating Mcl-1 expression Mcl-1 expression is regulated at multiple levels including regulation of transcription, modification of transcript, and post-translat ion modification (Fig. 14). The Mcl-1 protein has a very short half-life (171,172), and as such many of the primary means of regulation are dependent on tr anscriptional mechanisms. Physiological mechanisms governing Mcl-1 transcripti on are highly dependent on cell type and

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Extracellular Ligands Signal Transduction Pathways Transcription Factors Posttranscriptional Modification Posttranslational ModificationCytokines, GFs, CSFs, IFNs JAK/STAT, MEK/ERK, p38/MAPK, PI3K/AKT STAT3, PU.1, Elk-1 HIF-1 (hypoxia) E2F1 (cellular stress) Mcl-1 (antiapoptotic) Mcl-1S (proapoptotic) CaspaseCleavage: Mcl-1128-350(proapoptotic) Mcl-11-127(inactive) Phosphorylation: Thr-163 Ser-121/Thr-163 Ubiquitination Via Mule/ARFBP1 Figure 18. Mechanisms regulating Mcl-1. Mcl-1 is regulated in a context-dependent manner by transcriptional, post-transcrip tional, and post-translation mechanisms. Extracellular ligands signal through multiple transduction pathways to positively influence Mcl-1 transcription in part through STAT3, PU.1, and Elk-1. Hypoxia also directly upregulates Mcl-1 transcripti on through HIF-1, whereas stress-induced upregulation of E2F1 directly represses Mcl-1 transcription (italics: negative regulation ). Mcl-1 transcript may also be alternatively spliced to create a s horter form of Mcl-1 termed Mcl-1S, which is proapoptotic in na ture. Finally, Mcl-1 ma y be regulated posttranslationally through caspase cleavag e, phosphorylation, or ubiquitination. 69

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70 developmental state, and since much of the research on Mcl-1 has concentrated on hematopoietic tissues, many of the mechanisms regulating Mcl-1 transcription have been identified in that context. Transcription of Mcl-1 can be positively influenced by various extracellular stimuli including cytokines, grow th factors, colony stim ulating factors, and interferons (173,174). Depending on the stimuli, these signals are mediated by one or more transduction pathways including the JAK/STAT, MEK/ERK, p38/MAPK and PI3K/AKT signal transd uction pathways (173,174). Transcription factors residi ng at the ends of signal transduction pathways that positively regulate Mcl-1 transc ription work primarily through three response elements within the MCL-1 promoter. Induction of Mcl-1 transcription through the PI3K/AKT pathway is mediated through the cAMP res ponse element-binding (CREB) transcription factor, which directly binds a cAMP re sponse element (CRE)-2 upstream of the transcription start site (175). Activation via th e JAK/STAT pathway is the result of STAT binding to a serum-inducible element (SIE) (176-178), a nd activation through p38/MAPK-mediated pathways results in Ets family member PU.1 also binding to the SIE element (179). Additionally, activation through MEK/ERK also functions through the SIE element as mediated by Ets member Elk-1 (180). In additi on to transcriptional control mediated by signal transduction pa thways, Mcl-1 is also induced in hypoxic conditions through during binding of HIF-1 to the promoter (181). As previously eluded to, many of the previously described mechan isms regulating Mcl-1 transcription have been identified in hematopoietic tissue develo pment, and it is not clear what role these ligands and signal transduction pathways play in regulating Mcl-1 transciption in other tissues.

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71 While mechanisms positively regulating Mc l-1 transcription in the context of development and maturation of hematopoietic tissues are well described, less is known about how Mcl-1 is transcrip tionally regulated in other tissues and other contexts specifically how it is repressed during the induc tion of apoptosis. Given the lability of the Mcl-1 protein, it reasonable to hypothesize that transcriptional downregulation of Mcl-1 is not due to direct repressi on, but rather from a lack of positive signaling. Previous studies from our lab, however, have identified the E2F1 tran scription factor as directly binding to and repressing the MCL-1 promoter (122). Interestingly, this is independent of pRb family member binding, although the exac t mechanism has not yet been determined. As described in the following section, Mcl1 is thought to play a critical role in promoting the survival of malignant cells, and as such elucidating mechanisms regulating Mcl-1 transcription in this c ontext is of great importance. In addition to transcriptional regula tion, Mcl-1 is also subject to posttranscriptional and post-transl ational modification. In so me contexts, Mcl-1 undergoes alternative splicing to produce a shorter fr om of Mcl-1 (termed Mcl-1S) (182,183). This modification results in the loss of the TM do main as well as BH1-2 domains, giving way to an alternate Mcl-1 protein with a structur e similar to that of the BH3-only subfamily members (182,183). Indeed, Mcl-1S does not bind to proapoptotic Bcl-2 family members but instead binds to antiapoptotic members, and its overexpression is sufficient to induce apoptosis (182,183). Mechanisms of post-tran slational modification include cleavage, phosphorylation, and ubquitination. During apop tosis, Mcl-1 is subject to caspasemediated cleavage at conserve d aspartic acid residues ( 184,185), with one the resultant cleavage products being proapoptotic in nature in overexpr ession assays (184). In the

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72 case of phosphorylation, Mcl-1 is phosphorylat ed through multiple mechanisms at two specific serine residues that can either posit ively or negatively in fluence Mcl-1 protein levels. Oxidative stress inactivates Mcl-1 via JNK-mediated phosphor yalation at serine121 and threonine Thr-163 (186), and Mcl-1 is also phosphorylated at Thr-163 via TPA by Erk-dependent mechanismsleading to in creased protein stability (187,188). There are purported to be other phosphorylation sites in Mcl-1, but they have yet to be characterized (188). Finally, Mc l-1 is subject to ubiquitina tion by Mule/ARF-BP1, which negatively regulates Mcl-1 protein by targe ting it for proteasomal degradation (189). Taken together, Mcl-1 is subject to both positive and negative regulation at multiple levels through multiple mechanisms. Mcl-1 and human malignancy While in itself not a direct oncogene, as described in a previous section Mcl-1 may behave as an oncogene when present in combination with other oncogenic mutations due to its potent ability to promote surviv al. This is partially exemplified by studies demonstrating correlations between Mcl-1 ex pression and disease outcomes. In chronic lymphocytic leukemia, a higher level of Mcl1 expression correlates with failure to achieve complete remission, and in breast cancer Mcl-1 expression a ssociates with poor prognosis (190,191). Additionally, Mcl-1 expression is associated with disease progression in melanoma, and is also a predictor of surv ival in gastric carcinoma (192,193). Experiments utilizing siRNA-medi ated knockdown of Mcl-1 in cancerous cells has also pointed at the integral role Mcl-1 may play promoting malignant cell

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73 viability. Indeed, antisense de pletion of Mcl-1 in malignant cell can lead to decreased viability and the induction apoptosissuggestin g that interfering with Mcl-1 expression may prove to be a rational therapy for human malignancies (163-166). Summary and rationale Mcl-1 is an antiapoptotic Bcl-2 family me mber, and as such may act as a potent oncogene due to its ability to promote cell su rvival. Indeed, antisense depletion of Mcl-1 can induce loss of viability a nd apoptosis in malignant ce llsexemplifying the necessity of Mcl-1 expression to their su rvival (163-166). Since Mcl-1 is a labile protein, much of its regulation is thought to be dependent on transcriptional mechanisms. A publication from Moshynska et al. identified two novel sequence variants of the MCL-1 promoter within lymphocytes from chronic lymphoc ytic leukemia patients, but not within noncancerous tissue from the same individuals or in lymphocytes from 18 healthy control subjects (1). This result sugge sted that the variantsinserti ons of 6 or 18 nucleotides at position relative to the transcription start site ( 194)were CLL-related somatic oncogenic mutations. Moshynska et al. also determined that the 6and 18-nucleotide insertions were associated with elevated Mcl-1 expression, and proposed that the variant promoters could be used as a prognostic mark er. We independently identified and cloned the three observed sequence variants from can cer cell lines hereby referred to as the MCL-1 +0, +6 or +18 promoters. In contrast to Moshynska et al., we find the variant promoters to be identically present in bot h cancerous and adjacen t noncancerous clinical lung samples, suggesting that the variants are germ-line en coded. We also find the three

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74 variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that obs erved in cancerous cell lines. Furthermore, activity analysis of the thr ee variant promoters reveals th e MCL-1 +6 and +18 promoters to be less active than the MCL-1 +0 promot er, both during normal cellular homeostasis and under conditions that actively induce MC L-1 transcription. Given our results, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no represent a reliable prognostic marker for CLL.

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75 Experimental Procedures Promoter identification and screening Genomic DNA was extracted from cell lin es as described (Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001, p. 8.46-8.53). Mcl-1 promoter sequence from cell lines representative of the thr ee aberrances, namely H1299, K562, and T98G, was amplified from genomic DNA using a primer pair that spanned bases to (5'-AGG CCC GAG GTG CTC ATG GAA AGA-3') and +72 to +93 (5'-TTG AGG CCA AAC ATT GCC AGT CA-3') of what is referred to as the Mcl-1 +0 promoter. The resulting products were cloned into pCR2.1-TOPO using the TOPO TA Cloning Kit (Invitrogen) and the products sequenced by the Moffitt Molecular Biology Core. For larger scale screening purposes, a primer pair that spanned bases to (5'-AG C TTC CGG AGG GTT GCG CA-3') and to (5'-GGC ACT CAG AGC CTC CGA AGA-3') were used to amplify the Mcl-1 promoter with th e resulting products resolved on a 6% polyacrylamide gel and visualized af ter exposure to ethidium bromide.

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76 Cell lines Breast cancer cell lines screened consists of: MDA-MB-231, MDA-MB-361, MDA-MB-361, MDA-MB-435s, MDA-MB453, MDA-MB-468, MCF7, SK-BR-3 and T47D. Lung cancer lines screened co nsist of: H322, H358, H324, H661, H522, H146, H209, H417, H82 and H211. Paired clinical lung samples All patient and control donors provided informed consent as approved by the Institutional Review Board. Lung tumor a nd corresponding normal lung tissue specimens were collected from patients undergoing rout ine thoracotomy for su rgical resection of their malignancy. Resected specimens were briefly inspected by a su rgical pathologist and then snap-frozen in liquid nitrogen. Frozen sections were micros copically viewed to assess the proportion of tumor cells, normal cells and necrotic cells in tumor specimens to ensure absence of malignant cells in normal specimens. None of the patients had received radiation or chemothera py prior to sample collection. Healthy control samples Peripheral blood mononuclear cells we re collected from healthy normal volunteers. None of the volunteers had a known malignancy or illness. All were

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77 Caucasian except for one Hispanic and one Asia n volunteer. The ages ranged from 19 to 62, and 34 of the volunteers were female and 25 were male. Luciferase assays The above described pCR2.1-TOPO Mcl-1 +0, +6, and +18 constructs employed for the initial screening were shuttled into the pGL3 (Invitrogen) lu ciferase vectors, and the sequences were verified via sequencing by the Moffitt Molecular Biology Core. Luciferase assays were conducted using th e Dual-Luciferase Reporter Assay System (Promega) following the published protocol. NIH/3T3 and K562 cells were grown to ~70% confluency in 60 mm2 plates, and K562s were transfected at a density of 1.5 106 cells per 60 mm2 plate. Lipofectamine and PL US reagent (Invitrogen) were used for transfections following the published protocol All transfections were conducted in triplicate. DNA concentrations per transfection were as follows: 1 g pGL3/derivative, 0.1 g pRLTK internal control plasmid, and 0.9 g carrier DNA. Cells were washed once with phosphate-buffered saline (PBS) and give n new media at 4 hours after transfection. At 24 hours, cells were either harvested for an alysis or induced to differentiate with 100 nM phorbol 12-myristae 13-acetate (P MA). PMA treated cells were collected for analysis at 12 hours post treatment. For analysis of act ivity, luciferase activity was normalized to the internal renilla activity.

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78 Results Identification of two novel MCL-1 promoter variants An article entitled Prognostic Significance of a Short Sequence Insertion in the MCL-1 Promoter in Chronic Lymphocytic Leukemia identified two novel sequence variants of the Mcl-1 promot er within lymphocytes from chronic lymphocytic leukemia patients, but not within noncancerous tissue fr om the same individual or in lymphocytes from 18 healthy control subjects (1). This resu lt suggested that the va riantsinsertions of 6 and 18 nucleotides at position relative to the transcription start site as mapped by akgul et al. (194)were CLL -related somatic oncogenic mutations. Moshynska et al. also determined that the 6and 18-nucleotide insertions were associ ated with higher Mcl1 mRNA and protein, and may ther efore hold prognostic significance. In the course of analyzing the E2F1-med iated transcriptional repression of Mcl-1, we independently identified and cloned the three observed sequence variants from three human cancer cell lines, H1299(MCL-1 +0/+0) lung cancer cells, K562(MCL-1 +6/+6) erythroleukemia cells, and T98G(MCL-1 +0/+18) glioblastoma cells, representing the MCL-1 +0, +6, and +18 alleles, respectively. We next used polymerase chain reaction, followed by resolution of the PCR products on acrylam ide gels, to determine MCL-1 promoter status in a large number of cell lines and solid tumors. The MCL-1 +6 and MCL-1 +18

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79 promoters occurred with a re latively high frequency in ge nomic DNA derived from both breast and lung cancer cell line s, although the common MCL-1 +0 allele was the most prevalent (Table 1). The MCL-1 +6 and MCL-1 +18 promoter variants are not the result of somatic mutation We next wanted to determine if the variant promoters were somatic in origin. To address this issue, we analyzed the MCL1 promoter status of genomic DNA derived from 15 sets of paired lung cancer and adjacent normal lung tissue from patients undergoing routine thoracotomy for surgical resection of their malignancy. All samples were provided in deidentified fashion, and a ll patients provided informed consent as approved by the Institutional Review Board. In all 15 samples, the MCL-1 promoter profile was identical in cancerous and normal tissue (Fig. 18). Given this observation, we conclude that the variant prom oters are not somatic in origin and are germ-line encoded. The MCLl-1 +6 and MCL-1 +18 promoters are common polymorphisms While we established that the variant pr omoters were not the result of somatic mutation, we considered the possibility that the MCL-1 promoter variants may predispose to malignancy. To this end, we screened genomic DNA derived from 59 healthy individuals, all of whom provi ded informed consent, for the presence of the variant Mcl-1 promoters. Nearly half of the total alleles had one or both insertions, and the insertions occurred at frequencies similar to that obser ved in cancer cell lines (Fig. 19). Thus, it

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Table 1. The allelic frequencies of th e MCL-1 +0, MCL-1 +6, and MCL-1 +18 promoters in breast and lung cancer cell lines. MCL-1 +0 MCL-1 +6 MCL-1 +18 Allele Breast Lines (N=16)Lung Lines (N=20) 9 (56%) 10 (50%) 3 (19%) 4 (25%) 3 (15%) 7 (35%) MCL-1 +0 MCL-1 +6 MCL-1 +18 Allele Breast Lines (N=16)Lung Lines (N=20) 9 (56%) 10 (50%) 3 (19%) 4 (25%) 3 (15%) 7 (35%) +18 +6 +0 +18 +6 +0 Normal 1 Tumor 1 Normal 2 Tumor 2 Normal 3 Tumor 3 50 pbladder + Control No templatePaired Lung Biopsies MCL-1 +0 MCL-1 +6 MCL-1 +18 Allele Normal Lung (N=36)Lung Tumor (N=36) 25 (70%) 25 (70%) 5 (14%) 6 (16%) 5 (14%) 6 (16%)A B Figure 19. The variant MCL-1 promoters ar e not the result of somatic mutation. (A) Representative samples of the MCL-1 promoter status in genomic DNA from paired lung tumor biopsy and adjacent normal tissue de rived from patients undergoing routine thoracotomy as determined by resolving PCR products from the MCL-1 promoter on a polyacrylamide gel with subsequent visualizatio n. (B) The resultant allelic frequencies of all paired samples examined. 80

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+18 +6 +0 Healthy Donor DNA No template + Control 410100 410102 410107 410108 410111 410113 410116 410146 410154 410155 410158 410163 410164 410165 410097 50 bpladder 410046 410057 410063 410065 410069 410070 410071 410073 410074 410076 410077 410078 410086 410087 410024+18 +6 +050 bpladder No template + ControlHealthy Donor DNA MCL-1 +0 MCL-1 +6 MCL-1 +18 Allele Normal (N=118) 55 (47%) 24 (20%) 14 (33%)A B Figure 20. The variant MCL-1 promoters are prevalent in genomic DNA derived from healthy controls. (A) Representative samples of the MCL-1 promoter status in genomic DNA from healthy donor peripheral blood mononuclear cells as determined by resolving PCR products from the MCL-1 promoter on a polyacrylamide gel with subsequent visualization. (B) The resultant allelic frequencies of all healthy control samples examined. 81

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82 appears likely that the MCL-1 +6 and +18 pr omoter variants are likely common benign polymorphisms. The MCL-1 +6 and MCL-1 +18 promoters ar e less active than the common MCL-1 +0 promoter Mcl-1 belongs to the Bcl-1 family of pr oteins and may be a potent oncogene due to its ability to block apoptosis. Although we found the MCL-1 +6 and +18 polymorphisms to be quite common, we considered it possible that they could contribute to oncogenesis by rendering the promoter more active, thereby increasing the expression of MCL-1. To explore this po ssibility, we cloned the MCL1 +0, +6 and +18 promoters into a pGL3 luciferase vector, transfected the constructs into multiple cell lines, and determined promoter activity. Surprisingly the variant promoters displayed decreased activityboth during normal cellular homeos tasis and under conditions that actively induce Mcl-1 transcription (i .e., treatment with phorbol 12myristate 13-acetate (PMA)), with the +18 promoter displaying approxima tely half the activity of the MCL-1 +0 promoter (Fig. 20A, B and C). Taken together we conclude that the MCL-1 +6 and +18 variant promoters likely represent benign pol ymorphisms that probably do not represent a reliable prognostic marker.

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C 0 25 50 75 100 125 Construct:Relative Luciferase Activity no treatment 100 nMPMA MCF7 pGL3 hMCL -1 + 0 pGL3 hMCL 1 + 6 pGL3 hM C L 1 + 18B 0 50 100 150Relative Luciferase Activity no treatment 100 nMPMA K562 pGL3 hMCL -1 +0 pGL3 hMCL -1 +6 pGL3 -h M CL 1 +18Construct:A 0 100 200 300 400Relative Luciferase ActivityNIH/3T3 pGL3 hM C L -1 +0 pGL3 hM C L -1 + 6 pGL3 -hMCL 1 + 1 8Construct: Figure 21. The MCL-1 +6 and MCL-1 +18 pr omoters are less active than the MCL1 +0 promoter. (A) Luciferase constructs repr esenting the three variant MCL-1 promoters and a renilla control construct were cotransfected into NIH/3T3s with the MCL-1 promoter constructs assayed for activity relative to the renilla internal control 24 hours post transfection. (B and C) Transfec tions conducted as in figure 20A, except MCF7 and K562 cells were treated in para llel plus or minus PMA to induce Mcl-1 transcription. Cells were harves ted after 12 hours of PMA treatment. 83

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84 Discussion While there are significant disagreement s between our data and that published by Moshynska et al. (1), there is agreement as to the existence, location, and composition of the MCL-1 +6 and +18 variant promoters. Be yond that however, there is little common ground. Moshynska et al. claim that to have specifically found th e MCL-1 +6 and +18 promoters only in genomic DNA derived from lymphocytes from CLL patients and not within cancerous tissue derived from the sa me individuals or in lymphocytes from healthy control subjects, suggesting that the variants are CLL-related oncogenic mutations. In contrast, we find the variant promoters identically present in paired samples of cancerous and adjacent noncancerous lung, and also find the promoters prevalent in genomic DNA derived from healthy volunteers (195). Similar studie s investigating the variant MCL-1 promoters are in agreement w ith our results. Vargas et al. report the variant promoters present in 24 healthy control samples, and Iglesias-S erret et al. find the variant promoters present in lymphocytes from CLL patients as well as lymphocytes from 10 control subjects and mouth epithelial cells from 10 additional healthy control subjects (196,197). Experiments by Dicker et al. find MC L-1 promoter status identical between that observed in lymphocytes from CLL patients and in genomic DNA derived from buccal swabs, and further find the MCL-1 +6 and +18 promoters common DNA samples from health control individuals (198). Coenen et al. have also reported similar results

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85 (199). Given the bulk of evidence in support of our data, we c onfidently conclude that the variant MCL-1 promoters are not the result of a CLL-related oncogenic mutation and instead represent common benign polymorphisms. A second assertion from the Moshynska publ ication was that th e presence of the MCL-1 +6 and +18 promoters correlated wi th increased expre ssion of MCL-1 mRNA and protein (1). To investigate the effect of the MCL-1 +6 and +18 variants on promoter activity, we cloned the MCL-1 +0, +6, and +18 promoters in to luciferase vectors and assayed them for activity in multiple ce ll lines and found the MCL-1 +6 and +18 promoters to be less active than the co mmon MCL-1 +0 promot er, both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription (195). Unfortunately, most studies investigating th e variant MCL-1 promoters did not conduct expression assays, however one study did co mpare MCL-1 promoter status to Mcl-1 expression in CLL patients via microarray, but observed no correlation (198). It is possible that the reported positive correlati on by Moshynska et al. is real, however our promoter activity assay would argue otherwis e. Therefore, we a ssert that under our experimental conditions, the variant MCL-1 +6 and +18 promoters are less active than the common MCL-1 +0 promoter. Another major finding from the Moshynska et al. publication is that the MCL-1 +6 and +18 variant promoters positively co rrelate with risk of dying and decreased disease-free survival in CLL patients (1). Since our studies did not examine an association with CLL, we do not definitively disagree with this statement. However, the finding by ourselves and others that the MCL-1 +6 and +8 promoters are actually common polymorphisms with no discernable correlation to malignancy highlights a

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86 fundamental flaw in either th e screening technique, data reporting, or both employed by Moshynska et al. This in itself is suffici ent to be skeptical of any analyses and conclusions inferred from their data, yet other studies conducting similar sets of experiments have thoroughly demonstrated th at the MCL-1 +6 or +18 promoters do not correlate with disease outcomes in either CLL or acute lymphoblastic leukemia (ALL) (198-201). Thus, whether an error in the screenin g technique or an erro r in reporting, it is clear that the MCL-1 +6 and +18 promoter s hold no prognostic significance to CLL. It is unclear why there are so many disc repancies between the work of ourselves and others and that presented by Moshynska et al. As previously mentioned, the gross amounts of errors are likely th e result of a flawed screen ing technique, flawed data reporting, or both. In our experiments, we util ized a PCR-based screening technique that allowed for a clear distinction of both MCL-1 promoter alleles of a sample by virtue of differences in migration within the different sized PCR products. However, the technique employed by Moshynska et al. consisted of direct sequencing of PCR products, which in theory should be a reliable technique. And, gi ven the complete absence of the MCL-1 +6 and MCL-1 +18 promoters in every sample they examined except CLL cells, it is unlikely that this scenario could occur completely by chance. Taken together, we conclude that the MCL-1 +6 and +18 promoters are common benign polymorphisms that likely hold no prognostic value for CLL, that the MCL-1 +6 and +18 promoters are less active than the MCL-1 +0 promoter, and that the discrepancies found within the Moshynska publication are likely the resu lt of an error in data reporting.

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87 LIST OF REFERENCES 1. Moshynska, O., Sankaran, K., Pahw a, P., and Saxena, A. (2004) J Natl Cancer Inst 96(9), 673-682 2. Bracken, A. P., Ciro, M., Co cito, A., and Helin, K. (2004) Trends Biochem Sci 29(8), 409-417 3. DeGregori, J., and Johnson, D. G. (2006) Curr Mol Med 6(7), 739-748 4. Johnson, D. G., and Degregori, J. (2006) Curr Mol Med 6(7), 731-738 5. Cam, H., and Dynlacht, B. D. (2003) Cancer Cell 3(4), 311-316 6. Harbour, J. W., and Dean, D. C. (2000) Genes Dev 14(19), 2393-2409 7. Nevins, J. R. (2001) Hum Mol Genet 10(7), 699-703 8. DeGregori, J. (2002) Biochim Biophys Acta 1602 (2), 131-150 9. Dimova, D. K., and Dyson, N. J. (2005) Oncogene 24(17), 2810-2826 10. Dyson, N. (1998) Genes Dev 12(15), 2245-2262 11. Trimarchi, J. M., and Lees, J. A. (2002) Nat Rev Mol Cell Biol 3(1), 11-20 12. Wang, S., Ghosh, R. N., and Chellappan, S. P. (1998) Mol Cell Biol 18(12), 74877498 13. Kato, J., Matsushime, H., Hi ebert, S. W., Ewen, M. E., and Sherr, C. J. (1993) Genes Dev 7(3), 331-342 14. Meyerson, M., and Harlow, E. (1994) Mol Cell Biol 14(3), 2077-2086 15. Johnson, D. G., Ohtani, K., and Nevins, J. R. (1994) Genes Dev 8(13), 1514-1525

PAGE 102

88 16. Hsiao, K. M., McMahon, S. L., and Farnham, P. J. (1994) Genes Dev 8(13), 15261537 17. Neuman, E., Flemington, E. K., Sellers, W. R., and Kaelin, W. G., Jr. (1994) Mol Cell Biol 14 (10), 6607-6615 18. Ohtani, K., DeGregori, J ., and Nevins, J. R. (1995) Proc Natl Acad Sci U S A 92(26), 12146-12150 19. Dynlacht, B. D., Flores, O., Lees, J. A., and Harlow, E. (1994) Genes Dev 8(15), 1772-1786 20. DeGregori, J., Kowalik, T ., and Nevins, J. R. (1995) Molecular and cellular biology 15 (8), 4215-4224 21. Zhang, L., and Wang, C. (2006) Oncogene 25(18), 2615-2627 22. Xu, M., Sheppard, K. A., Peng, C. Y., Y ee, A. S., and Piwnica-Worms, H. (1994) Mol Cell Biol 14(12), 8420-8431 23. Marti, A., Wirbelauer, C., Scheffner, M., and Krek, W. (1999) Nat Cell Biol 1(1), 14-19 24. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M., Albert, D. M., and Dryja, T. P. (1986) Nature 323(6089), 643-646 25. Knudson, A. G., Jr. (1971) Proc Natl Acad Sci U S A 68(4), 820-823 26. Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366(6456), 704-707 27. Veltman, J. A., Fridlyand, J., Pejavar, S., Olshen, A. B., Korkola, J. E., DeVries, S., Carroll, P., Kuo, W. L., Pinkel, D., Albertson, D., Cordon-Cardo, C., Jain, A. N., and Waldman, F. M. (2003) Cancer Res 63(11), 2872-2880

PAGE 103

89 28. Orlic, M., Spencer, C. E., Wang, L., and Gallie, B. L. (2006) Genes Chromosomes Cancer 45(1), 72-82 29. Oeggerli, M., Tomovska, S., Schraml, P., Calvano-Forte, D., Schafroth, S., Simon, R., Gasser, T., Mihatsch, M. J., and Sauter, G. (2004) Oncogene 23(33), 5616-5623 30. Foster, C. S., Falconer, A., Dodson, A. R., Norman, A. R., Dennis, N., Fletcher, A., Southgate, C., Dowe, A., Dearnaley, D., Jhavar, S., Eeles, R., Feber, A., and Cooper, C. S. (2004) Oncogene 23(35), 5871-5879 31. Grasemann, C., Gratias, S., Stephan, H., Schuler, A., Schramm, A., Klein-Hitpass, L., Rieder, H., Schneider, S., Kappes, F ., Eggert, A., and Lohmann, D. R. (2005) Oncogene 24(42), 6441-6449 32. Saito, M., Helin, K., Valentine, M. B., Gr iffith, B. B., Willman, C. L., Harlow, E., and Look, A. T. (1995) Genomics 25(1), 130-138 33. Fujita, Y., Sakakura, C., Shimomura, K., Nakanishi, M., Yasuoka, R., Aragane, H., Hagiwara, A., Abe, T., Inazawa, J., and Yamagishi, H. (2003) Hepatogastroenterology 50(54), 1857-1863 34. Brookman-Amissah, N., Duchesnes, C., Williamson, M. P., Wang, Q., Ahmed, A., Feneley, M. R., Mackay, A., Freeman, A., Fenwick, K., Iravani, M., Weber, B., Ashworth, A., and Masters, J. R. (2005) Prostate cancer and prostatic diseases 8 (4), 335-343 35. Watanabe, T., Imoto, I., Katahira, T., Hirasawa, A., Ishiwata, I., Emi, M., Takayama, M., Sato, A., and Inazawa, J. (2002) Jpn J Cancer Res 93(10), 11141122

PAGE 104

90 36. Postma, C., Hermsen, M. A., Coffa, J., B aak, J. P., Mueller, J. D., Mueller, E., Bethke, B., Schouten, J. P., Stolte M., and Meijer, G. A. (2005) J Pathol 205(4), 514-521 37. Suzuki, T., Yasui, W., Yokozaki, H., Naka, K., Ishikawa, T., and Tahara, E. (1999) Int J Cancer 81(4), 535-538 38. Lee, T. A., and Farnham, P. J. (2000) Oncogene 19(18), 2257-2268 39. Melillo, R. M., Helin, K., Lowy, D. R., and Schiller, J. T. (1994) Mol Cell Biol 14(12), 8241-8249 40. Pierce, A. M., Schneider-Broussard, R., Gimenez-Conti, I. B., Russell, J. L., Conti, C. J., and Johnson, D. G. (1999) Mol Cell Biol 19(9), 6408-6414 41. Russell, J. L., Weaks, R. L., Be rton, T. R., and Johnson, D. G. (2006) Oncogene 25(6), 867-876 42. Frame, F. M., Rogoff, H. A., Pickering, M. T., Cress, W. D., and Kowalik, T. F. (2006) Oncogene 25(23), 3258-3266 43. Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000) Mol Cell Biol 20(1), 273-285 44. Lomazzi, M., Moroni, M. C., Jensen, M. R., Frittoli, E., and Helin, K. (2002) Nat Genet 31(2), 190-194 45. Lazzerini Denchi, E., Attwooll, C., Pasini, D., and Helin, K. (2005) Mol Cell Biol 25(7), 2660-2672 46. Yee, A. S., Reichel, R., Kovesdi, I., and Nevins, J. R. (1987) Embo J 6(7), 20612068

PAGE 105

91 47. Ivey-Hoyle, M., Conroy, R., Huber, H. E., Goodhart, P. J., Oliff, A., and Heimbrook, D. C. (1993) Mol Cell Biol 13(12), 7802-7812 48. Lees, J. A., Saito, M., Vidal, M., Vale ntine, M., Look, T., Harlow, E., Dyson, N., and Helin, K. (1993) Mol Cell Biol 13(12), 7813-7825 49. Leone, G., Nuckolls, F., Ishida, S., Adam s, M., Sears, R., Jakoi, L., Miron, A., and Nevins, J. R. (2000) Mol Cell Biol 20(10), 3626-3632 50. He, Y., Armanious, M. K., Thomas, M. J., and Cress, W. D. (2000) Oncogene 19(30), 3422-3433 51. Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z. X., Xu, G., Wydner, K. L., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev 8(22), 2665-2679 52. Beijersbergen, R. L., Kerkhoven, R. M., Zhu, L., Carlee, L., Voorhoeve, P. M., and Bernards, R. (1994) Genes Dev 8(22), 2680-2690 53. Buck, V., Allen, K. E., Sorensen, T., Bybee, A., Hijmans, E. M., Voorhoeve, P. M., Bernards, R., and La Thangue, N. B. (1995) Oncogene 11(1), 31-38 54. Itoh, A., Levinson, S. F., Morita, T., Kour embanas, S., Brody, J. S., and Mitsialis, S. A. (1995) Cell Mol Biol Res 41(3), 147-154 55. Trimarchi, J. M., Fairchild, B., Verona, R., Moberg, K., Andon, N., and Lees, J. A. (1998) Proc Natl Acad Sci U S A 95(6), 2850-2855 56. Cartwright, P., Muller, H., Wagener, C., Holm, K., and Helin, K. (1998) Oncogene 17(5), 611-623 57. Gaubatz, S., Wood, J. G., a nd Livingston, D. M. (1998) Proc Natl Acad Sci U S A 95(16), 9190-9195

PAGE 106

92 58. de Bruin, A., Maiti, B., Jakoi, L., Timme rs, C., Buerki, R., and Leone, G. (2003) J Biol Chem 278(43), 42041-42049 59. Di Stefano, L., Jensen, M. R., and Helin, K. (2003) Embo J 22(23), 6289-6298 60. Logan, N., Delavaine, L., Graham, A ., Reilly, C., Wilson, J., Brummelkamp, T. R., Hijmans, E. M., Bernards, R., and La Thangue, N. B. (2004) Oncogene 23(30), 5138-5150 61. Maiti, B., Li, J., de Bruin, A., Gordon, F., Timmers, C., Opavsky, R., Patil, K., Tuttle, J., Cleghorn, W., and Leone, G. (2005) J Biol Chem 280(18), 18211-18220 62. Christensen, J., Cloos, P., Toftegaard, U., Klinkenberg, D., Bracken, A. P., Trinh, E., Heeran, M., Di Stefano, L., and Helin, K. (2005) Nucleic Acids Res 33(17), 5458-5470 63. Sears, R., Ohtani, K., and Nevins, J. R. (1997) Mol Cell Biol 17(9), 5227-5235 64. Trimarchi, J. M., Fairchild, B., Wen, J., and Lees, J. A. (2001) Proc Natl Acad Sci U S A 98(4), 1519-1524 65. Logan, N., Graham, A., Zhao, X., Fish er, R., Maiti, B., Leone, G., and La Thangue, N. B. (2005) Oncogene 24(31), 5000-5004 66. Johnson, D. G., Schwarz, J. K., Cress, W. D., and Nevins, J. R. (1993) Nature 365(6444), 349-352 67. Qin, X. Q., Livingston, D. M., Kaelin, W. G., Jr., and Adams, P. D. (1994) Proc Natl Acad Sci U S A 91(23), 10918-10922 68. DeGregori, J., Leone, G., Miron, A., Jakoi, L., and Nevins, J. R. (1997) Proc Natl Acad Sci U S A 94(14), 7245-7250

PAGE 107

93 69. Lukas, J., Petersen, B. O., Holm, K., Bartek, J., and Helin, K. (1996) Mol Cell Biol 16(3), 1047-1057 70. DeGregori, J., Leone, G., Ohtani, K ., Miron, A., and Nevins, J. R. (1995) Genes Dev 9(23), 2873-2887 71. Schwarz, J. K., Bassing, C. H., Kovesdi, I., Datto, M. B., Blazing, M., George, S., Wang, X. F., and Nevins, J. R. (1995) Proc Natl Acad Sci U S A 92(2), 483-487 72. Mann, D. J., and Jones, N. C. (1996) Curr Biol 6 (4), 474-483 73. Johnson, D. G., Cress, W. D., Jakoi, L., and Nevins, J. R. (1994) Proc Natl Acad Sci U S A 91(26), 12823-12827 74. Shan, B., and Lee, W. H. (1994) Mol Cell Biol 14 (12), 8166-8173 75. Singh, P., Wong, S. H., and Hong, W. (1994) Embo J 13(14), 3329-3338 76. Xu, G., Livingston, D. M., and Krek, W. (1995) Proc Natl Acad Sci U S A 92(5), 1357-1361 77. Chen, Q., Liang, D., Yang, T., Leone, G., and Overbeek, P. A. (2004) Dev Neurosci 26 (5-6), 435-445 78. Chen, Q., Hung, F. C., Fromm, L., and Overbeek, P. A. (2000) Invest Ophthalmol Vis Sci 41 (13), 4223-4231 79. Guy, C. T., Zhou, W., Kaufman, S., and Robinson, M. O. (1996) Mol Cell Biol 16(2), 685-693 80. Pierce, A. M., Fisher, S. M., Con ti, C. J., and Johnson, D. G. (1998) Oncogene 16(10), 1267-1276 81. Paulson, Q. X., McArthur, M. J., and Johnson, D. G. (2006) Cell Cycle 5(2), 184190

PAGE 108

94 82. Scheijen, B., Bronk, M., van der Meer, T ., De Jong, D., and Bernards, R. (2004) J Biol Chem 279(11), 10476-10483 83. Conner, E. A., Lemmer, E. R., Omori, M., Wirth, P. J., Factor, V. M., and Thorgeirsson, S. S. (2000) Oncogene 19(44), 5054-5062 84. Agger, K., Santoni-Rugiu, E., Holmberg, C., Karlstrom, O., and Helin, K. (2005) Oncogene 24(5), 780-789 85. Pierce, A. M., Gimenez-Conti, I. B., Schneider-Broussard, R., Martinez, L. A., Conti, C. J., and Johnson, D. G. (1998) Proc Natl Acad Sci U S A 95(15), 88588863 86. Vigo, E., Muller, H., Prosperini, E., Hate boer, G., Cartwright, P., Moroni, M. C., and Helin, K. (1999) Mol Cell Biol 19(9), 6379-6395 87. Kowalik, T. F., DeGregori, J., Leone, G., Jakoi, L., and Nevins, J. R. (1998) Cell Growth Differ 9(2), 113-118 88. Wu, X., and Levine, A. J. (1994) Proc Natl Acad Sci U S A 91(9), 3602-3606 89. Kowalik, T. F., DeGregori, J., Schwar z, J. K., and Nevins, J. R. (1995) Journal of virology 69(4), 2491-2500 90. Holmberg, C., Helin, K., Seheste d, M., and Karlstrom, O. (1998) Oncogene 17(2), 143-155 91. Lazzerini Denchi, E., and Helin, K. (2005) EMBO Rep 6(7), 661-668 92. Berton, T. R., Mitchell, D. L., Guo, R., and Johnson, D. G. (2005) Oncogene 24(15), 2449-2460

PAGE 109

95 93. Wikonkal, N. M., Remenyik, E., Knezevic D., Zhang, W., Liu, M., Zhao, H., Berton, T. R., Johnson, D. G., and Brash, D. E. (2003) Nat Cell Biol 5(7), 655660 94. Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N. J. (1996) Cell 85(4), 537-548 95. Cloud, J. E., Rogers, C., Reza, T. L., Ziebold, U., Stone, J. R., Picard, M. H., Caron, A. M., Bronson, R. T., and Lees, J. A. (2002) Mol Cell Biol 22(8), 26632672 96. Zhu, J. W., Field, S. J., Gore, L., Thom pson, M., Yang, H., Fujiwara, Y., Cardiff, R. D., Greenberg, M., Orkin, S. H., and DeGregori, J. (2001) Mol Cell Biol 21(24), 8547-8564 97. Murga, M., Fernandez-Capetillo, O., Fi eld, S. J., Moreno, B., Borlado, L. R., Fujiwara, Y., Balomenos, D., Vicario, A., Carrera, A. C., Orkin, S. H., Greenberg, M. E., and Zubiaga, A. M. (2001) Immunity 15(6), 959-970 98. DeRyckere, D., and DeGregori, J. (2005) J Immunol 175(2), 647-655 99. Li, F. X., Zhu, J. W., Hogan, C. J., and DeGregori, J. (2003) Mol Cell Biol 23(10), 3607-3622 100. Stevaux, O., and Dyson, N. J. (2002) Curr Opin Cell Biol 14(6), 684-691 101. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and Nevins, J. R. (2001) Mol Cell Biol 21(14), 4684-4699 102. Ren, B., Cam, H., Takahashi, Y., Volk ert, T., Terragni, J., Young, R. A., and Dynlacht, B. D. (2002) Genes Dev 16 (2), 245-256

PAGE 110

96 103. Weinmann, A. S., Yan, P. S., Oberley, M. J., Huang, T. H., and Farnham, P. J. (2002) Genes Dev 16(2), 235-244 104. Zhu, W., Giangrande, P. H., and Nevins, J. R. (2004) Embo J 23(23), 4615-4626 105. Hernando, E., Nahle, Z., Juan, G., Diaz-Rodriguez, E., Alaminos, M., Hemann, M., Michel, L., Mittal, V., Gerald, W., Benezra, R., Lowe, S. W., and CordonCardo, C. (2004) Nature 430(7001), 797-802 106. Hiebert, S. W., Packham, G., Strom, D. K., Haffner, R., Oren, M., Zambetti, G., and Cleveland, J. L. (1995) Mol Cell Biol 15(12), 6864-6874 107. Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden, K. H. (1998) Nature 395(6698), 124-125 108. Kamijo, T., Weber, J. D., Zambetti, G., Zindy, F., Roussel, M. F., and Sherr, C. J. (1998) Proc Natl Acad Sci U S A 95(14), 8292-8297 109. Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998) Cell 92 (6), 725-734 110. Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997) Nature 387(6630), 296-299 111. Kubbutat, M. H., Jones, S. N., and Vousden, K. H. (1997) Nature 387(6630), 299303 112. Canman, C. E., Lim, D. S., Cimprich, K. A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M. B., and Siliciano, J. D. (1998) Science 281(5383), 16771679 113. Banin, S., Moyal, L., Shieh, S., Ta ya, Y., Anderson, C. W., Chessa, L., Smorodinsky, N. I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. (1998) Science 281(5383), 1674-1677

PAGE 111

97 114. Irwin, M., Marin, M. C., Phillips, A. C., Seelan, R. S., Smith, D. I., Liu, W., Flores, E. R., Tsai, K. Y., Jacks, T., V ousden, K. H., and Kaelin, W. G., Jr. (2000) Nature 407(6804), 645-648 115. Stiewe, T., and Putzer, B. M. (2000) Nat Genet 26 (4), 464-469 116. Hershko, T., Chaussepied, M., Oren, M., and Ginsberg, D. (2005) Cell Death Differ 12 (4), 377-383 117. Real, P. J., Sanz, C., Gutierrez, O., Pipaon, C., Zubiaga, A. M., and FernandezLuna, J. L. (2006) FEBS Lett 580(25), 5905-5909 118. Rodriguez, J. M., Glozak, M. A., Ma, Y., and Cress, W. D. (2006) J Biol Chem 281(32), 22729-22735 119. Moroni, M. C., Hickman, E. S., Lazzerin i Denchi, E., Caprara, G., Colli, E., Cecconi, F., Muller, H., and Helin, K. (2001) Nat Cell Biol 3(6), 552-558 120. Xie, W., Jiang, P., Miao, L., Zhao, Y., Zhimin, Z., Qing, L., Zhu, W. G., and Wu, M. (2006) Nucleic Acids Res 34(7), 2046-2055 121. Nahle, Z., Polakoff, J., Davuluri, R. V., McCurrach, M. E., Jacobson, M. D., Narita, M., Zhang, M. Q., Lazebnik, Y., Bar-Sagi, D., and Lowe, S. W. (2002) Nat Cell Biol 4(11), 859-864 122. Croxton, R., Ma, Y., Song, L., Haura, E. B., and Cress, W. D. (2002) Oncogene 21(9), 1359-1369 123. Crowe, D. L., Nguyen, D. C., Tsang, K. J., and Kyo, S. (2001) Nucleic Acids Res 29(13), 2789-2794 124. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269(5229), 1424-1427

PAGE 112

98 125. Ma, Y., and Cress, W. D. (2007) Oncogene 26(24), 3532-3540 126. Wang, C., Hou, X., Mohapatra, S., Ma, Y., Cress, W. D., Pledger, W. J., and Chen, J. (2005) The Journal of biological chemistry 280(13), 12339-12343 127. Radhakrishnan, S. K., Feliciano, C. S., Na jmabadi, F., Haegebarth, A., Kandel, E. S., Tyner, A. L., and Gartel, A. L. (2004) Oncogene 23(23), 4173-4176 128. Stevens, C., and La Thangue, N. B. (2004) DNA Repair (Amst) 3(8-9), 1071-1079 129. Ramos, S., Khademi, F., Somes h, B. P., and Rivero, F. (2002) Gene 298(2), 147157 130. Lundgren, R., Mandahl, N., Heim, S., Lim on, J., Henrikson, H., and Mitelman, F. (1992) Genes Chromosomes Cancer 4(1), 16-24 131. Emi, M., Fujiwara, Y., Nakajima, T., Tsuchiya, E., Tsuda, H., Hirohashi, S., Maeda, Y., Tsuruta, K., Miyaki M., and Nakamura, Y. (1992) Cancer Res 52(19), 5368-5372 132. Bova, G. S., Carter, B. S., Bussemakers, M. J., Emi, M., Fujiwara, Y., Kyprianou, N., Jacobs, S. C., Robinson, J. C., Epstei n, J. I., Walsh, P. C. and et al. (1993) Cancer Res 53(17), 3869-3873 133. Fujiwara, Y., Emi, M., Ohata, H., Kato, Y., Nakajima, T., Mori, T., and Nakamura, Y. (1993) Cancer Res 53 (5), 1172-1174 134. Sunwoo, J. B., Holt, M. S., Radford, D. M., Deeker, C., and Scholnick, S. B. (1996) Genes Chromosomes Cancer 16(3), 164-169 135. Brown, M. R., Chuaqui, R., Vocke, C. D., Berchuck, A., Middleton, L. P., Emmert-Buck, M. R., and Kohn, E. C. (1999) Gynecol Oncol 74(1), 98-102

PAGE 113

99 136. Wistuba, II, Behrens, C., Virmani, A. K., Milchgrub, S., Syed, S., Lam, S., Mackay, B., Minna, J. D., and Gazdar, A. F. (1999) Cancer Res 59(8), 1973-1979 137. Rivero, F., Dislich, H., Glockner, G., and Noegel, A. A. (2001) Nucleic Acids Res 29(5), 1068-1079 138. Chang, F. K., Sato, N., Kobayashi-Simorowski, N., Yoshihara, T., Meth, J. L., and Hamaguchi, M. (2006) J Mol Biol 364(3), 302-308 139. Collins, T., Stone, J. R., and Williams, A. J. (2001) Mol Cell Biol 21(11), 36093615 140. Furukawa, M., He, Y. J., Bo rchers, C., and Xiong, Y. (2003) Nat Cell Biol 5(11), 1001-1007 141. Geyer, R., Wee, S., Anderson, S., Yates, J., and Wolf, D. A. (2003) Mol Cell 12(3), 783-790 142. Pintard, L., Willis, J. H., Willems, A. Johnson, J. L., Srayko, M., Kurz, T., Glaser, S., Mains, P. E., Tyers, M., Bowerman, B., and Peter, M. (2003) Nature 425(6955), 311-316 143. Xu, L., Wei, Y., Reboul, J., Vaglio, P., Sh in, T. H., Vidal, M., Elledge, S. J., and Harper, J. W. (2003) Nature 425(6955), 316-321 144. Wilkins, A., Ping, Q., and Carpenter, C. L. (2004) Genes Dev 18(8), 856-861 145. St-Pierre, B., Jiang, Z., Egan, S. E., and Zacksenhaus, E. (2004) Gene Expr Patterns 5 (2), 245-251 146. Hamaguchi, M., Meth, J. L., von Klitzing, C., Wei, W., Esposito, D., Rodgers, L., Walsh, T., Welcsh, P., King, M. C., and Wigler, M. H. (2002) Proc Natl Acad Sci U S A 99(21), 13647-13652

PAGE 114

100 147. Yoshihara, T., Collado, D., and Hamaguchi, M. (2007) Biochem Biophys Res Commun 358 (4), 1076-1079 148. Siripurapu, V., Meth, J., Kobayash i, N., and Hamaguchi, M. (2005) J Mol Biol 346(1), 83-89 149. Wang, C., Chen, L., Hou, X., Li, Z., Kabra, N., Ma, Y., Nemoto, S., Finkel, T., Gu, W., Cress, W. D., and Chen, J. (2006) Nature cell biology 8(9), 1025-1031 150. Ma, Y., Cress, W. D., and Haura, E. B. (2003) Molecular cancer therapeutics 2(1), 73-81 151. Kalejta, R. F., Shenk, T., and Beavis, A. J. (1997) Cytometry 29 (4), 286-291 152. Cory, S., Huang, D. C., and Adams, J. M. (2003) Oncogene 22(53), 8590-8607 153. Adams, J. M., and Cory, S. (2007) Oncogene 26(9), 1324-1337 154. Zou, H., Henzel, W. J., Liu, X ., Lutschg, A., and Wang, X. (1997) Cell 90(3), 405-413 155. Li, P., Nijhawan, D., Budiha rdjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91(4), 479-489 156. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J. (2001) Science 292(5517), 727-730 157. Reynolds, J. E., Li, J., Craig, R. W., and Eastman, A. (1996) Exp Cell Res 225(2), 430-436 158. Reynolds, J. E., Yang, T., Qian, L., Jenkinson, J. D., Zhou, P., Eastman, A., and Craig, R. W. (1994) Cancer Res 54(24), 6348-6352

PAGE 115

101 159. Zhou, P., Qian, L., Kozopas, K. M., and Craig, R. W. (1997) Blood 89(2), 630643 160. Zhou, P., Levy, N. B., Xie, H., Qian, L., Lee, C. Y., Gascoyne, R. D., and Craig, R. W. (2001) Blood 97(12), 3902-3909 161. Zhou, P., Qian, L., Bieszczad, C. K., Noelle, R., Binder, M., Levy, N. B., and Craig, R. W. (1998) Blood 92(9), 3226-3239 162. Matsushita, K., Okita, H., Suzuki, A ., Shimoda, K., Fukuma, M., Yamada, T., Urano, F., Honda, T., Sano, M., Iwanaga, S., Ogawa, S., Hata, J., and Umezawa, A. (2003) Mol Cell Endocrinol 203 (1-2), 105-116 163. Moulding, D. A., Giles, R. V., Spiller, D. G., White, M. R., Tidd, D. M., and Edwards, S. W. (2000) Blood 96(5), 1756-1763 164. Zhang, B., Gojo, I., and Fenton, R. G. (2002) Blood 99(6), 1885-1893 165. Derenne, S., Monia, B., Dean, N. M., Ta ylor, J. K., Rapp, M. J., Harousseau, J. L., Bataille, R., and Amiot, M. (2002) Blood 100(1), 194-199 166. Leuenroth, S. J., Grutkoski, P. S., Ayala, A., and Simms, H. H. (2000) J Leukoc Biol 68(1), 158-166 167. Rinkenberger, J. L., Horning, S., Klocke, B., Roth, K., and Korsmeyer, S. J. (2000) Genes Dev 14(1), 23-27 168. Opferman, J. T., Letai, A., Beard, C ., Sorcinelli, M. D., Ong, C. C., and Korsmeyer, S. J. (2003) Nature 426(6967), 671-676 169. Opferman, J. T., Iwasaki, H., Ong, C. C., Suh, H., Mizuno, S., Akashi, K., and Korsmeyer, S. J. (2005) Science 307 (5712), 1101-1104 170. Dzhagalov, I., St John, A., and He, Y. W. (2007) Blood 109(4), 1620-1626

PAGE 116

102 171. Chao, J. R., Wang, J. M., Lee, S. F., Peng, H. W., Lin, Y. H., Chou, C. H., Li, J. C., Huang, H. M., Chou, C. K., Kuo, M. L., Yen, J. J., and Yang-Yen, H. F. (1998) Mol Cell Biol 18 (8), 4883-4898 172. Nijhawan, D., Fang, M., Traer, E., Z hong, Q., Gao, W., Du, F., and Wang, X. (2003) Genes Dev 17(12), 1475-1486 173. Craig, R. W. (2002) Leukemia 16(4), 444-454 174. Le Gouill, S., Podar, K., Harousseau, J. L., and Anderson, K. C. (2004) Cell Cycle 3(10), 1259-1262 175. Wang, J. M., Chao, J. R., Chen, W., Kuo, M. L., Yen, J. J., and Yang-Yen, H. F. (1999) Mol Cell Biol 19 (9), 6195-6206 176. Puthier, D., Bataille, R., and Amiot, M. (1999) Eur J Immunol 29(12), 3945-3950 177. Epling-Burnette, P. K., Liu, J. H., Catle tt-Falcone, R., Turkson, J., Oshiro, M., Kothapalli, R., Li, Y., Wang, J. M., Yang-Yen, H. F., Karras, J., Jove, R., and Loughran, T. P., Jr. (2001) J Clin Invest 107(3), 351-362 178. Epling-Burnette, P. K., Zhong, B., Bai, F., Jiang, K., Bailey, R. D., Garcia, R., Jove, R., Djeu, J. Y., Loughran, T. P., Jr., and Wei, S. (2001) J Immunol 166(12), 7486-7495 179. Wang, J. M., Lai, M. Z., and Yang-Yen, H. F. (2003) Mol Cell Biol 23(6), 18961909 180. Townsend, K. J., Zhou, P., Qian, L., Bieszczad, C. K., Lowrey, C. H., Yen, A., and Craig, R. W. (1999) J Biol Chem 274(3), 1801-1813 181. Piret, J. P., Minet, E., Cosse, J. P ., Ninane, N., Debacq, C., Raes, M., and Michiels, C. (2005) J Biol Chem 280(10), 9336-9344

PAGE 117

103 182. Bae, J., Leo, C. P., Hsu, S. Y., and Hsueh, A. J. (2000) J Biol Chem 275(33), 25255-25261 183. Bingle, C. D., Craig, R. W., Swales, B. M., Singleton, V., Zhou, P., and Whyte, M. K. (2000) J Biol Chem 275(29), 22136-22146 184. Michels, J., O'Neill, J. W., Dallman, C. L., Mouzakiti, A., Habens, F., Brimmell, M., Zhang, K. Y., Craig, R. W., Marcuss on, E. G., Johnson, P. W., and Packham, G. (2004) Oncogene 23(28), 4818-4827 185. Clohessy, J. G., Zhuang, J., and Brady, H. J. (2004) Br J Haematol 125(5), 655665 186. Inoshita, S., Takeda, K., Hatai, T., Terada, Y., Sano, M., Hata, J., Umezawa, A., and Ichijo, H. (2002) J Biol Chem 277(46), 43730-43734 187. Domina, A. M., Smith, J. H., and Craig, R. W. (2000) J Biol Chem 275(28), 21688-21694 188. Domina, A. M., Vrana, J. A., Gregor y, M. A., Hann, S. R., and Craig, R. W. (2004) Oncogene 23(31), 5301-5315 189. Zhong, Q., Gao, W., Du, F., and Wang, X. (2005) Cell 121 (7), 1085-1095 190. Kitada, S., Andersen, J., Akar, S., Zapa ta, J. M., Takayama, S., Krajewski, S., Wang, H. G., Zhang, X., Bullrich, F., Croce, C. M., Rai, K., Hines, J., and Reed, J. C. (1998) Blood 91(9), 3379-3389 191. Ding, Q., He, X., Xia, W., Hsu, J. M., Chen, C. T., Li, L. Y., Lee, D. F., Yang, J. Y., Xie, X., Liu, J. C., and Hung, M. C. (2007) Cancer Res 67 (10), 4564-4571 192. Zhuang, L., Lee, C. S., Scolyer, R. A., McCarthy, S. W., Zhang, X. D., Thompson, J. F., and Hersey, P. (2007) Mod Pathol 20(4), 416-426

PAGE 118

104 193. Maeta, Y., Tsujitani, S., Matsumoto, S., Yamaguchi, K., Tatebe, S., Kondo, A., Ikeguchi, M., and Kaibara, N. (2004) Gastric Cancer 7(2), 78-84 194. Akgul, C., Turner, P. C., White, M. R., and Edwards, S. W. (2000) Cell Mol Life Sci 57(4), 684-691 195. Freeman, S. N., Bepler, G., Haura, E., Sutphen, R., and Cress, W. D. (2005) J Natl Cancer Inst 97(14), 1088-1089; aut hor reply 1093-1085 196. Vargas, R. L., Felgar, R. E ., and Rothberg, P. G. (2005) J Natl Cancer Inst 97(14), 1089-1090; author reply 1093-1085 197. Iglesias-Serret, D., Coll-Mulet, L., Santidrian, A. F., Navarro-Sabate, A., Domingo, A., Pons, G., and Gil, J. (2005) J Natl Cancer Inst 97(14), 1090-1091; author reply 1093-1095 198. Dicker, F., Rauhut, S., Kohlmann, A., Ke rn, W., Schoch, C., Haferlach, T., and Schnittger, S. (2005) J Natl Cancer Inst 97(14), 1092-1093; au thor reply 10931095 199. Coenen, S., Pickering, B., Potter, K. N., Johnson, P. W., Stevenson, F. K., and Packham, G. (2005) Haematologica 90(9), 1285-1286 200. Tobin, G., Skogsberg, A., Thunberg, U., Laurell, A., Aleskog, A., Merup, M., Sundstrom, C., Roos, G., Nilsson, K., and Rosenquist, R. (2005) Leukemia 19(5), 871-873 201. Nenning, U. C., Eckert, C., Wellmann, S., Barth, A., Henze, G., and Seeger, K. (2005) J Natl Cancer Inst 97(14), 1091-1092; author reply 1093-1095

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ABOUT THE AUTHOR Scott N. Freeman received his B.S. degree Cum Laude with a major in Biology and a minor in Chemistry from Central Mi chigan University in Mount Pleasant, Michigan in 2002. Scott N. Freeman continue d his education by accepting a position in the Cancer Biology Ph.D. program at the University of South Florida where he conducted his graduate research under the supervision of W. Douglas Cress, Ph.D. at the H. Lee Moffitt Cancer Center and Research Institute. Scott N. Freeman has one first author publication in The Journal of the National Cancer Institute a second author publication in Cancer Biology and Therapy and at the time of this publication, has a first author publication pending for The Journal of Biological Chemistry


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Analysis of E2F1 target genes involved in cell cycle and apoptosis
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ABSTRACT: One of the main results of Rb-E2F pathway disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the induction of apoptosis. Given the potential negative biological effects associated with deregulated E2F activity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct physiological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregulated during mitosis due in part to E2F1, and that overexpression of RhoBTB2 increases the S-phase fraction and slows the rate of proliferation. We also find RhoBTB2 similarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis.^ Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL-1---MCL-1 +6 and +18---as initially published by Moshynska et al (1). In contrast to Moshynska et al., we find the variant promoters identically present in both cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that observed in cancerous cell lines. In further contrast, we find the activity of the MCL-1 +6 and +18 promoters approximately 50% less than the common MCL-1 +0 promoter---both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription.^ Given our results and those of others, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no [sic] represent a reliable prognostic marker for CLL as reported by Moshynska et al.
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