Bcl-2 family members regulate the sensitivity to 2-deoxy-D-glucose in lymphomas

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1 University of Iowa Iowa Research Online Theses and Dissertations Fall 2011 Bcl-2 family members regulate the sensitivity to 2-deoxy-D-glucose in lymphomas Oksana Zagorodna University of Iowa Copyright 2011 Oksana Zagorodna This dissertation is available at Iowa Research Online: Recommended Citation Zagorodna, Oksana. "Bcl-2 family members regulate the sensitivity to 2-deoxy-D-glucose in lymphomas." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Other Biochemistry, Biophysics, and Structural Biology Commons

2 BCL-2 FAMILY MEMBERS REGULATE THE SENSITIVITY TO 2-DEOXY-D-GLUCOSE IN LYMPHOMAS by Oksana Zagorodna An Abstract Of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Free Radical and Radiation Biology in the Graduate College of The University of Iowa December 2011 Thesis Supervisor: Associate Professor C. Michael Knudson

3 1 ABSTRACT Bcl-2 family members are important regulators of apoptosis, and their altered expression is often involved in oncogenesis. Of particular importance are the levels of Bcl-2 family members in forming lymphomas. We studied two groups of murine thymic T cell lymphomas derived from either Bcl-2 or Bax overexpression in order to predict their sensitivity and resistance to treatments. While the growth rate and histological characteristics were similar for both lymphoma groups, Bax-derived lymphomas failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2-derived lymphomas often halted proliferation following radiation delivery and rarely had p53 mutations. Bax-derived lymphomas were uniformly sensitive to treatment with 2-deoxy-D-glucose (2DG) while all Bcl-2-derived lymphomas were resistant. This led us to hypothesize that the Bcl-2 family is involved in 2DG-induced cell death. Focusing on the mechanism of 2DG toxicity in Bax-derived lymphomas, our studies demonstrate the following: cell death involved the activation of proapoptotic Bax, was effectively blocked by anti-apoptotic Bcl-2, and was mediated, at least in part, by the BH3-only family member Bim. Based on these results, we explored whether a BH3 mimetic (ABT-737) could sensitize lymphomas to 2DG killing. Indeed, a combination of ABT-737 with 2DG enhanced killing in Bax-derived lymphomas and resensitized Bcl-2- overexpressing lymphomas to 2DG. Since both 2DG and BH3 mimetics are currently in clinical trials, understanding their killing mechanisms and optimal combinations are of potential clinical significance. The work in this dissertation demonstrates a novel role of Bcl-2 family member proteins in regulating 2DG toxicity and may predict response to

4 2 2DG treatment. The information found presents a new strategy of combining 2DG with BH3 mimetics to improve existing lymphoma therapies. Abstract Approved: Thesis Supervisor Title and Department Date

5 BCL-2 FAMILY MEMBERS REGULATE THE SENSITIVITY TO 2-DEOXY-D-GLUCOSE IN LYMPHOMAS by Oksana Zagorodna A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Free Radical and Radiation Biology in the Graduate College of The University of Iowa December 2011 Thesis Supervisor: Associate Professor C. Michael Knudson

6 Copyright by OKSANA ZAGORODNA 2011 All Rights Reserved

7 Graduate College The University of Iowa Iowa City, Iowa This is to certify that the Ph.D. thesis of CERTIFICATE OF APPROVAL PH.D. THESIS Oksana Zagorodna has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Free Radical and Radiation Biology at the December 2011 graduation. Thesis Committee: C. Michael Knudson, Thesis Supervisor Michael B. Cohen Prabhat C. Goswami Dawn E. Quelle Douglas R. Spitz

8 To my family with my deepest gratitude for their unwavering love and support. ii

9 ACKNOWLEDGEMENTS I would like to thank Dr. C. Michael Knudson for his guidance and mentorship throughout my time of working on a graduate degree in his lab. I am grateful for the numerous scientific discussions, within the scope of our work, as well as outside the field of our expertise. They added to shaping my view of science and to my maturation as a researcher. I would like to thank all the members of my thesis committee: Dr. Michael Cohen, Dr. Prabhat Goswami, Dr. Dawn Quelle, and Dr. Douglas Spitz for their critical insights on my work in its development, and for their continuous help and willingness to discuss scientific matters whenever I asked. I would also like to thank Dr. Larry Oberley for his invaluable impact on establishing a collaborative environment within the Free Radical and Radiation Biology Program. Aside from science, I have enjoyed working in the Knudson Lab thanks to a wonderful group of people, past and present lab members, who make every day better: Sean Martin, Sih-han Wang, Chris Van de Wetering, Agshin Taghiyev, Peter Harris, Agnieszka Wydra, Michael Moriarty, and Robyn Blendowski. It has been great working with you guys. iii

10 ABSTRACT Bcl-2 family members are important regulators of apoptosis, and their altered expression is often involved in oncogenesis. Of particular importance are the levels of Bcl-2 family members in forming lymphomas. We studied two groups of murine thymic T cell lymphomas derived from either Bcl-2 or Bax overexpression in order to predict their sensitivity and resistance to treatments. While the growth rate and histological characteristics were similar for both lymphoma groups, Bax-derived lymphomas failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2-derived lymphomas often halted proliferation following radiation delivery and rarely had p53 mutations. Bax-derived lymphomas were uniformly sensitive to treatment with 2-deoxy-D-glucose (2DG) while all Bcl-2-derived lymphomas were resistant. This led us to hypothesize that the Bcl-2 family is involved in 2DG-induced cell death. Focusing on the mechanism of 2DG toxicity in Bax-derived lymphomas, our studies demonstrate the following: cell death involved the activation of proapoptotic Bax, was effectively blocked by anti-apoptotic Bcl-2, and was mediated, at least in part, by the BH3-only family member Bim. Based on these results, we explored whether a BH3 mimetic (ABT-737) could sensitize lymphomas to 2DG killing. Indeed, a combination of ABT-737 with 2DG enhanced killing in Bax-derived lymphomas and resensitized Bcl-2- overexpressing lymphomas to 2DG. Since both 2DG and BH3 mimetics are currently in clinical trials, understanding their killing mechanisms and optimal combinations are of potential clinical significance. The work in this dissertation demonstrates a novel role of Bcl-2 family member proteins in regulating 2DG toxicity and may predict response to iv

11 2DG treatment. The information found presents a new strategy of combining 2DG with BH3 mimetics to improve existing lymphoma therapies. v

12 TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS viii ix xi CHAPTER I. INTRODUCTION 1 Bcl-2 family proteins are critical regulators of apoptosis 1 Bcl-2 family proteins regulate mitochondrial outer membrane permeabilization (MOMP) 2 BH3-only proteins activate Bax/Bak in two ways 4 The antiapoptotic family member, Bcl-2, and its involvement in oncogenesis 4 The proapoptotic family member, Bax, and its involvement in oncogenesis 5 The Bcl-2 family and functional p53 6 BH3-only members and oncogenesis 7 BH3 mimetics for cancer therapy 9 Altered metabolism in cancer 10 2-Deoxy-D-glucose (2DG) in cancer therapy 11 A link between Bcl-2 proteins and ER stress 12 Purpose of the study 13 CHAPTER II. T CELL LYMPHOMA (TCL) CELL LINES DERIVED FROM EITHER BAX OR BCL-2 OVEREPXPRESSION IN MICE DIFFER IN THEIR SENSITIVITY TO 2-DEOXY-D-GLUCOSE 21 Summary 21 Introduction 21 Materials and Methods 23 Results 27 Discussion 31 CHAPTER III. 2-DEOXY-D-GLUCOSE-INDUCED TOXICITY IS REGULATED BY BCL-2 FAMILY MEMBERS AND IS ENHANCED BY ANTAGONIZING BCL-2 IN LYMPHOMA CELL LINES 52 Summary 52 Introduction 53 Materials and Methods 56 Results 61 Discussion 66 vi

13 CHAPTER IV. DISCUSSION AND FUTURE DIRECTIONS 105 Extending the outcome of 2DG studies from murine lymphomas to human malignancies may assist with improving treatments 105 2DG upregulates Bim in more than one way 106 CHOP/GADD153 may be necessary for Bim upregulation and 2DG toxicity 107 ER stress-induced cell death pathways are regulated by Bcl-2 family proteins 108 Targeting cancer by inhibiting UPR signaling 109 Targeting Bcl-2 family members in cancer therapy 113 Cancer therapy will become personalized 116 REFERENCES 117 vii

14 LIST OF TABLES Table 1. Doubling times in TCLs are comparable. 36 Table 2. Summary of responses in Bax and Bcl-2 TCLs following treatments. 50 viii

15 LIST OF FIGURES Figure 1. The Bcl-2 family of apoptotic regulators. 15 Figure 2. Models of the Bcl-2 family members interactions. 17 Figure 3. Chemical structures of D-glucose, D-mannose, and 2-deoxy-D-glucose. 19 Figure 4. Histological examples of thymic tumors from Bax 38/1 and Bcl-2 p 27-/- mice. 34 Figure 5. Viability of Bax TCLs and Bcl-2 TCLs varies following ionizing radiation (IR). 38 Figure 6. Total cell counts in Bax TCLs versus Bcl-2 TCLs differ following ionizing radiation (IR). 40 Figure 7. Altered cell cycle progression in Bax TCLs versus Bcl-2 TCLs differs following ionizing radiation. 42 Figure 8. P53 protein levels in Bax versus Bcl-2 TCLs vary following ionizing radiation (IR). 44 Figure 9. Sequencing results of p53 demonstrate missense mutations. 46 Figure 10. Viability differs in Bax and Bcl-2 TCLs following 2DG treatment. 48 Figure 11. TCLs are uniformly sensitive to 2DG treatment. 71 Figure 12. Caspase 3 and PARP are cleaved following 2DG treatment. 73 Figure 13. Pharmacologic inhibition of caspases does not block caspase 3 cleavage following 2DG treatment. 75 Figure 14. Bax is activated following 2DG treatment. 77 Figure 15. Bcl-2 overexpression in TCLs protects from 2DG toxicity. 79 Figure 16. Bcl-2 expression protects clonogenic abilities of TCLs following 2DG treatment. 81 Figure 17. Bax activation is inhibited in TCL-Bcl-2 lines following 2DG treatment. 83 Figure 18. ATP levels are decreased following 2DG treatment in TCLs. 85 ix

16 Figure 19. ABT-737, a Bcl-2 antagonist, restores sensitivity to 2DG in TCL-Bcl-2 lines. 87 Figure 20. ABT-737, a Bcl-2 antagonist induces sensitivity to 2DG in TCLs. 89 Figure 21. ABT-737 inhibits 2DG-caused binding of Bim and Bmf BH3-only proteins to Bcl Figure 22. Downregulation of Bim protein inhibits 2DG toxicity. 93 Figure 23. Mannose delays 2DG-induced toxicity in TCLs. 95 Figure 24. 2DG upregulates ER stress and UPR target genes. 97 Figure 25. Bim upregulation occurs by ER stress dependent pathway. 99 Figure 26. Bim upregulation occurs by ER stress dependent and independent pathways. 101 Figure 27. B cell lymphoma cell lines are sensitive to 2DG treatment. 103 x

17 LIST OF ABBREVIATIONS 17AAG: 2DG: 2DG6P 17-allylamino-17-demethoxygeldanamycin 2-Deoxy-D-glucose 2-Deoxy-D-glucose-6-Phosphate APAF-1 Apoptotic protease-activating factor 1 ATF6 Activating transcription factor 6 Bax TCL T cell lymphoma derived from Bax overexpressing mice Bcl-2 TCL T cell lymphoma derived from Bcl-2 overexpressing mice (p27 -/-) BH Bcl-2 homology BH3 Bcl-2 homology 3 BrdU CICD CHOP CMML DOC DNA ECL ER ERAD FACS FAS GADD153 GAPDH Bromodeoxyuridine Caspase-independent cell death C/EBP homologous protein; also GADD153 Chronic myelomonocytic leukemia Deoxycholate Deoxyribonucleic acid Enhanced chemiluminescence Endoplasmic reticulum ER-associated protein degradation Fluorescence-activated cell sorting Fatty acid synthase Growth arrest- and DNA damage-inducible gene 153; also CHOP Glyceraldehyde-3-Phosphate Dehydrogenase GRP78 Glucose-regulated protein 78 xi

18 H&E HSP IMM IR Hematoxylin and eosin Heat shock protein Inner mitochondrial membrane Ionizing radiation IRE1 Inositol-requiring enzyme 1 MEFs MOMP NP40 OMM PARP PERK PBS PCR PI PUMA SDS SMAC TCL TCL-Bcl-2 UPR UVP Murine embryonic fibrobasts Mitochondrial outer membrane permeabilization Nonyl phenoxypolyethoxylethanol Outer mitochondrial membrane Poly(ADP-ribose) polymerase PKR-like ER kinase Phosphate buffered saline Polymerase chain reaction Propidium iodide p53 upregulated modulator of apoptosis Sodium dodecyl sulfate Second mitochondria-derived activator of caspase T cell lymphoma T cell lymphoma line engineered to overexpress Bcl-2 Unfolded protein response Ultra-violet products xii

19 1 CHAPTER I INTRODUCTION Bcl-2 family proteins are critical regulators of apoptosis Apoptosis is a process of cell death that eliminates damaged or unwanted cells and is essential for normal development, homeostasis and immune responses regulation. Apoptosis was first reported by Kerr, Wyllie and Currie, who recognized that this process is active and inherently programmed (Kerr et al., 1972). Perturbation in the regulation of apoptosis contributes to many diseases, including cancer. The characteristic apoptotic cellular changes at a biochemical and morphological levels include loss of the plasma membrane asymmetry, which exposes phosphatidyl serine from the inner to the outer leaflet, membrane blebbing, cell shrinkage chromatin condensation, and deoxyribonucleic acid (DNA) fragmentation. These changes are induced by a group of proteases called caspases that are divided in two types: initiator and effector (executioner) caspases. During apoptosis, protein substrates in the cell are cleaved by effector caspases (3, 6, and 7). Effector caspases are cleaved by initiator caspases (2, 8, and 9). Initiator caspases are activated either by death receptor engagement (extrinsic pathway) or APAF-1. APAF-1 is activated by proteins that are released from the intermembrane space of mitochondria, such as cytochrome c and SMAC (intrinsic pathway) (Scaffidi et al., 1998). This process of mitochondrial outer membrane permeabilization, termed MOMP (see the following section), is regulated by Bcl-2 family proteins that share Bcl-2 homology (BH) domains (Fig. 1). The BH3 domain is common to all Bcl-2 family members and is known to be

20 2 essential for their function. The Bcl-2 family of genes can be broadly divided into an antiapoptotic group (such as Bcl-2, Bcl-X L, Bcl-W, Mcl-1) and a proapoptotic group based on their function in cell death. The proapoptotic members can be further divided into multidomain members (such as Bax and Bak) and the BH3-only subfamily members (such as Bim, Bad, Bmf) (Youle and Strasser, 2008). Bcl-2 family proteins regulate mitochondrial outer membrane permeabilization (MOMP) In a cell-free system developed using extracts from the frog eggs, the fraction enriched with mitochondria was required for apoptosis (Newmeyer et al., 1994). Further research identified cytochrome c as the factor that caused caspase activation (Liu et al., 1996) and placed mitochondria in the center of apoptosis pathways. It is well established that Bcl-2 proteins regulate apoptosis by controlling the permeability of the mitochondrial outer membrane (Chipuk and Green, 2008; Kuwana et al., 2002). Proapoptotic Bcl-2 family proteins lead to mitochondrial outer membrane permeabilization (MOMP), while antiapoptotic members inhibit MOMP. MOMP is an event generally considered to be the point of no return during apoptosis (Chipuk et al., 2006; Chipuk and Green, 2008). Following MOMP, proteins (e.g., cytochrome c) normally found between the outer and inner mitochondrial membranes (OMM, IMM), are released into the cytosol. The release of the proteins from the intermembrane mitochondrial space does not occur when the multidomain proapoptotic proteins Bax and Bak are absent (Wei et al., 2000). These molecules are inserted into the outer membrane of mitochondria and oligomerized during MOMP (Antignani and Youle, 2006; Antonsson et al., 2000; Korsmeyer et al.,

21 3 2000). Thus, the activation of Bax and Bak is considered an essential gateway for mitochondrial events leading to apoptosis. In support of this, cells deficient in Bax and Bak demonstrated resistance to multiple apoptotic stimuli, such as application of ultraviolet radiation, growth factor deprivation, treatment with etoposide, and treatment with the endoplasmic reticulum (ER) stress inducer tunicamycin (Wei et al., 2001). Furthermore, in vivo studies registered that while mice deficient in either Bax or Bak had a mild phenotype, Bax/Bak double deficient mice developed excessive cell numbers in hematopoietic and central nervous systems, interdigital webs, and imperforate vaginal canals in females (Lindsten et al., 2000). Upon activation, Bax and Bak are known to undergo conformational change, which can be detected by conformation specific antibodies. Recently, the interaction between the Bim BH3 domain and Bax has been characterized (Gavathiotis et al., 2008). Transient interactions led to a conformational change of Bax/Bak resulting in the exposure of the BH3 domain of Bax/Bak and induced homo-dimer or oligomer formation (Gavathiotis et al., 2008). In order to convert from its inactive cytosolic form to its active membrane bound form, Bax was shown to undergo an N-terminal conformational change (Upton et al., 2007) and localize to the outer mitochondrial membrane in a manner dependent upon its C-terminus. It is not clear at which point of these Bax/Bak changes MOMP occurs. Upon MOMP, if the activation of caspases is impaired, cells can still die through caspase-independent cell death (CICD) (Cheng et al., 2001). The precise mechanism of CICD remains elusive; however, redox perturbations and energy loss have been suggested to be involved during the release of intermembrane proteins (Chipuk and Green, 2005).

22 4 BH3-only proteins activate Bax/Bak in two ways The genetic pathways of apoptosis were characterized in C. elegans (Conradt and Horvitz, 1998), and the BH3-only protein homologue, Egl-1, is upstream of the Bcl-2 homologue, Ced-9 (there is no Bax/Bak homologue in C. elegans). Likewise, the BH3- only proteins in mammals act upstream of the multidomain Bcl-2 family members (Huang and Strasser, 2000). Two models have been put forward as to how BH3-only proteins interact with the multi-domain members. In the direct activation model (Chipuk and Green, 2008; Kuwana et al., 2005; Letai et al., 2002), Bax and Bak are activated by select BH3-only members that have the ability to activate Bax/Bak directly (Bim, Bid, Puma, Bmf, Noxa), while inhibition by antiapoptotic proteins is removed by derepressor/sensitizer BH3-only proteins (Fig. 2A) (Du et al., 2011; Kuwana et al., 2005; Letai et al., 2002; Ren et al., 2010). The neutralization model predicts that BH3-only proteins bind and inhibit antiapoptotic Bcl-2 proteins, leading to the activation of Bax/Bak (Willis et al., 2007). Although inhibiting the antiapoptotic repertoire is essential to MOMP and cell death, MOMP can be executed most efficiently when both direct activation and neutralization models are involved (Du et al., 2011; Merino et al., 2009). The antiapoptotic family member Bcl-2 and its involvement in oncogenesis The Bcl-2 (B-cell lymphoma 2) gene was found in the immunoglobulin heavy chain locus as the result of the chromosomal translocation t(14;18) in human follicular B cell lymphoma (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto et al., 1985). The oncogenic activity of Bcl-2 is distinct from other oncogenes that stimulate cell proliferation (Beier et al., 2000) in that Bcl-2 induces oncogenesis by inhibiting cell

23 5 death. Further, it is known that if Bcl-2 is expressed together with other oncogenes, such as c-myc, tumor formation is greatly enhanced (Vaux et al., 1988). The induction in tumorigenesis is believed to be due to Bcl-2-caused inhibition of apoptosis and subsequent cell survival. While many studies show that higher Bcl-2 levels are associated with tumor development, some reports suggest that this is not always the case. For example, higher Bcl-2 expression correlated with improved survival in colon, breast cancer and lymphoma patients (Buglioni et al., 1999; Callagy et al., 2006; Rosenwald et al., 2002). Bcl-2 overexpression has been associated with normal chromosome number (van de Wetering et al., 2007). Furthermore, overexpression of Bcl-2 has been reported to delay hepatocellular carcinoma formation in c-myc transgenic mice (de La Coste et al., 1999). The paradoxical inhibitory effect of Bcl-2 on tumorigenesis could be due to its complex role in suppressing apoptosis and cell proliferation. The disparate effects of Bcl-2 on tumor formation could therefore be due to studying these effects in different systems. The proapoptotic family member Bax and its involvement in oncogenesis Other Bcl-2 family members, such as Bax, have also been shown to play a role in oncogenesis. A number of studies associate the loss of function of Bax with oncogenesis (Eischen et al., 2001; Miyashita and Reed, 1995; Yin et al., 1997). Bax was shown to be inactivated in human colon cancer by somatic frameshift mutations (Rampino et al., 1997), in gastrointestinal cancers by missense mutations (Gil et al., 1999), and high Bax expression was associated with better prognosis of disease-free and overall survival in

24 6 patients with acute myeloid leukemia (Ong et al., 2000). This indicates the importance of Bax in suppressing oncogenesis. Despite the evidence of suppressing oncogenesis, higher Bax levels are sometimes found to be associated with a higher rate of relapse in childhood acute lymphocytic leukemia (Hogarth and Hall, 1999) and with poor outcome in acute myeloid leukemia (Kohler et al., 2002). We found that transgenic mice overexpressing Bax,under the Lck promoter, developed T cell lymphomas (Knudson et al., 2001). These mice have decreased thymic cellularity, presumably because of Bax overexpression, prior to developing lymphomas. We speculate that cells that have acquired survival mechanisms against higher levels of Bax grew and became malignant eventually. The lymphomas in the patients reported to have high Bax levels might have resulted from the similar process. These findings suggest that Bcl-2 family members may have a role in oncogenesis that is distinct from their regular apoptotic functions, or that excessive apoptosis may be a contributor to oncogenesis. The Bcl-2 family and functional p53 The paradoxical effects of Bcl-2 family members in oncogenesis are not fully understood, yet their relationship with p53 may be involved in explaining the different effects observed in cancer. The tumor suppressor protein, p53, has a powerful role of conserving genomic stability. Among performing many other functions, p53 interacts with the Bcl-2 family members (Chipuk et al., 2004). While it is known that p53 is frequently mutated and becomes dysfunctional in various cancers, the pressure of selecting for p53 deficiency in oncogenesis appears to be alleviated when apoptotic pathways are inactivated (Eischen et al., 2001; Miyashita and Reed, 1995; Yin et al.,

25 7 1997). Indeed, tumor cell variants overexpressing Bcl-2 or suppressing p53 showed rapid expansion in growing tumors. However, no accumulations of p53-deficient variants were observed in tumors formed by the cells overexpressing Bcl-2, indicating that p53 deficiency lacks selective advantage when Bcl-2 is overexpressed. Since p53 deficiency was demonstrated to predispose to aneuploid tumor formation, the alleviation of selecting for p53 deficiency in the Bcl-2 overexpressing environment restricts the expansion of genetically unstable cells, therefore delaying tumor progression (Gurova et al., 2002; Harvey et al., 1993). This could partly explain the earlier described higher survival in patients who have tumors overexpressing Bcl-2. BH3-only members and oncogenesis BH3-only molecules are known to respond to different types of stimuli and undergo activation in diverse ways (Shibue and Taniguchi, 2006). The activation mechanisms are complex and include various transcriptional pathways and posttranslational modifications, many of which could be altered in cancer, thereby increasing chances of BH3-only abnormal activation/regulation in cancer cells. Downregulation of BH3-only members has been observed in carcinogenesis, and it is hypothesized that BH3-only members have a function of tumor suppressors. Studies on single or double knockout mice for various BH3-only members (Bid, Bim, Puma) demonstrate potentiation of tumor formation and resistance to apoptotic treatments. For example, Bid-deficient mice were shown to develop clonal malignancy closely resembling chronic myelomonocytic leukemia (CMML) later in their life (Zinkel et al., 2003). The loss of Bim was shown to dramatically increase the rate of Myc-induced tumorigenesis, and losing one bim allele was as effective as losing both alleles (Egle et al., 2004).

26 8 Interestingly, while the p19arf/p53 pathway is frequently mutated in Bim+/+ Eµ-Myc mice, no deregulation of this pathway was seen in most Bim-deficient tumors, indicating Bim reduction is an effective alternative to dysfunctional p53 pathway. Recently, loss of Bmf was shown to accelerate the development of gamma irradiation-induced thymic lymphomas in mice, indicative of its tumor suppressor function in lymphoma (Labi et al., 2008). Together, studies in mice suggest the involvement of BH3-only members in oncogenesis. Studies in human cancers rarely show genetic mutations in BH3-only genes, but frequent genetic and epigenetic silencing of the BH3-only genes has been reported. For example, homozygous deletions of BIM were found in mantle cell lymphomas and in cell lines derived from patients with B cell Non-Hodgkin s lymphomas (Mestre-Escorihuela et al., 2007; Tagawa et al., 2005). Hypermethylation in BIM promoter region occurs in Burkitt s lymphoma cell lines and patient biopsies, further supporting the involvement of BH3-only members in cancer (Mestre-Escorihuela et al., 2007). MicroRNAs, known to be involved in tumorigenesis, were shown to target and downregulate gene expression in BH3-only members Bim and Bmf (Gramantieri et al., 2009; Terasawa et al., 2009). The prognostic value of Bim and Puma was also shown as a strong independent predictor of disease-free and overall survival in human colon carcinoma (Sinicrope et al., 2008). Cumulatively, the expression levels of BH3 members in cancer cells vary and affect both anti- and pro- apoptotic function. Recently, a BH3 profiling strategy has been developed to help identify the apoptotic effects in cancer cells and predict their sensitivities to treatments (Certo et al., 2006; Deng et al., 2007). For BH3 profiling, mitochondria are extracted and exposed to a series of BH3 peptides to monitor their effects on MOMP, as

27 9 measured by cytochrome c release (Certo et al., 2006; Deng et al., 2007). Examining the pattern of cytochrome c release in response to BH3 peptides identifies the antiapoptotic proteins used to maintain cancer cell survival, which helps with identifying the targets in cancer treatment. BH3 mimetics for cancer therapy One of the hallmarks acquired by cancer cells to survive is the ability to evade apoptosis (Hanahan and Weinberg, 2011). The regulation of apoptotic processes is accomplished with the involvement of Bcl-2 family members. The abnormal governance of apoptotic events by the Bcl-2 family can contribute to carcinogenesis. Blocking the antiapoptotic Bcl-2 family members is recognized as an important strategy on the way to improving cancer therapy. Recent progress in structural studies at atomic levels on Bcl-2 family proteins has led to the development of drugs that mimic the function of the BH3 domains through which Bcl-2 members interact (Nguyen et al., 2007; Oltersdorf et al., 2005). Specifically, the structure of the complex between the peptide from the BH3 region of BH3 proteins and the prosurvival Bcl-2 protein is informative (Sattler et al., 1997). A number of BH3 mimetic compounds have recently been developed (Marzo and Naval, 2008). BH3 mimetics are identified from screenings of chemical compounds that show high affinity to binding Bcl-X L. These compounds are further tailored by the use of structural analysis to increase their affinity (Oltersdorf et al., 2005). ABT-737 was one of the first BH3 mimetic molecules synthesized (Oltersdorf et al., 2005). The design originated from nuclear magnetic resonance screening through a chemical library to identify small molecules with binding efficiency to the hydrophobic groove of Bcl-X L. Further medicinal chemistry application of techniques such as parallel

28 10 synthesis and structure-based design led to producing ABT-737. This molecule has high affinity for Bcl-2, Bcl-X L, and Bcl-W, and is the first compound closely resembling BH3- only protein affinities, in particular the binding profile of Bad-BH3 (Lee et al., 2007; Oltersdorf et al., 2005; van Delft et al., 2006). ABT-737 exhibits apoptotic effects when applied to tumor cells (lung cancer, lymphoma, and lymphoblastic leukemia) (Oltersdorf et al., 2005; Shoemaker et al., 2006; van Delft et al., 2006), is able to potentiate killing when combined with other drugs (Kang et al., 2007), and was shown to be effective in tumor xenograft models (Konopleva et al., 2006). ABT-263 (Navitoclax) is a newer generation compound and an oral analog for ABT-737 that is in clinical trials for treating a variety of malignancies (Tse et al., 2008) ( May 2011). Altered metabolism in cancer In order for a normal cell to become neoplastic, it must change and acquire a number of characteristics, or hallmarks, of the cancer phenotype. This includes selfsufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replication, sustained angiogenesis, and evading apoptosis (Hanahan and Weinberg, 2011). Recently, deregulated cellular energetics has been recognized as an emerging hallmark of cancer (Hanahan and Weinberg, 2011). The deregulated cellular energetics in cancer cells involves increased glycolysis and reduced oxidative phosphorylation even under aerobic conditions; a phenomenon known as aerobic glycolysis {{203 Warburg 1956;}}. The reasons for this difference in metabolism between normal versus cancer cells remain obscure. One thought is that cancer cells exist in an altered redox environment, and increasing glycolysis increases the production of reducing equivalents to maintain redox balance and support cell proliferation (Spitz et

29 11 al., 2000). Another theory suggests that the adaptation in cancer cell metabolism is due to facilitating the greater uptake and conversion of nutrients necessary to produce more cells (Vander Heiden et al., 2009). Yet another thought adds that cancer cells consume more glucose than normal counterparts to maintain sufficient ATP supply (Pelicano et al., 2006). Irrespective of which of these models is correct, it is clear that cancer cells take up and consume glucose at higher rates than their non-transformed counterparts. This unique property of cancer cells provides an opportunity for the treatment of cancer. 2-Deoxy-D-glucose (2DG) in cancer therapy 2-Deoxy-D-glucose (2DG) is a long-studied drug in cancer therapy. It has been explored as a glucose analog and is taken up by cells in the same manner as D-glucose (Fig. 3). After uptake, 2DG is converted to 2-deoxy-D-glucose-6-Phospate (2DG6P) by hexokinase, and this product inhibits glucose phosphoisomerase as well as affects the downstream glycolytic enzymes {{31 Crane 1954; 216 Nirenberg 1958; 42 Tower 1958;}}. Additionally, 2DG can inhibit the metabolism of glucose in the pentose phosphate pathways (Aykin-Burns et al., 2009). 2DG treatment leads to metabolic stress and cell death with greater selectivity for cancer cells versus normal cells (Aykin-Burns et al., 2009; Liu et al., 2001). It was shown to reach above 5 mm concentration in human blood and was well tolerated {{103 Mohanti 1996;}}. Aside from acting as a glycolytic inhibitor, 2DG inhibits N-linked glycosylation by acting as a competitive inhibitor of mannose addition to carbohydrate chains. The ability of 2DG to inhibit glycosylation should have been anticipated when one recognizes that 2-deoxy-D-mannose (2DM) and 2-deoxy-D-glucose are actually the same compound (Fig. 3). 2DG inhibition of glycosylation leads to perturbations in proper protein folding

30 12 and ER stress that activates the unfolded protein response (UPR). The affects of 2DG on N-linked glycosylation and ER stress can be reversed by exogenous addition of mannose (Datema and Schwarz, 1978; Kurtoglu et al., 2007). Persistent ER stress and UPR activate complex signaling pathways and may lead to cell death. One of these pathways involves the upregulation of CHOP/GADD153, a transcription factor that alone can activate a wide variety of downstream target genes (Xu et al., 2005; Zinszner et al., 1998). Studies in cells and animals deficient in GADD153 indicate that GADD153 is of particular importance in ER stress-induced cell death (Oyadomari and Mori, 2004). Applying 2DG to select breast and pancreatic cancer cell lines demonstrates the perturbation of N-linked glycosylation and induction of cell death that can be blocked by mannose addition (Kurtoglu et al., 2007). Combined, these results demonstrate that 2DG causes cell death by two distinct pathways: inhibition of glycolysis and inhibition of N- linked glycosylation. A link between Bcl-2 proteins and ER stress Although the understanding of ER stress-induced cell death pathway is still incomplete, evidence points at links to the Bcl-2 family members. For example, ER stress activates Bim and Puma, BH3-only members of the Bcl-2 family (Galehdar et al., 2010; Puthalakath et al., 2007). At least one of the activation mechanisms of Bim was by CHOP/GADD153-mediated direct transcriptional induction. Importantly, deficiency in Bim or Puma demonstrated protection from ER stress-induced apoptosis both in vivo and in vitro (Puthalakath et al., 2007; Reimertz et al., 2003). Cumulatively, findings to date exhibit a link between UPR and members of the Bcl-2 family leading to apoptotic cell death (Szegezdi et al., 2009).

31 13 Purpose of the study The broad objective of this thesis project is to examine the potential utility of 2DG in lymphoid malignancies with a goal of improving the existing treatments. Tumor cell lines involved in the studies were derived from models of B cell and T cell lymphomas to determine their relative sensitivity to 2DG. B cell neoplasms were generated as a result of Myc induction (Han et al., 2006). T cell lymphoma models were generated as a result of manipulations in Bcl-2 family members. One group of T cell lymphoma lines was derived from Lck-Bcl-2 transgenic mice lacking p27. These mice have elevated Bcl-2 levels in T cells and are chromosomally stable (Unpublished, (Cheng et al., 2008)). Another group of T cell lymphoma lines was derived from Lck-Bax38/1 transgenic mice. These mice have high levels of Bax in T cells, show increased chromosome instability and have evidence of oxidative stress (van de Wetering et al., 2008). Tumor development and chromosome instability in this model are inhibited by Bcl-2 (Luke et al., 2003; van de Wetering et al., 2007) and by SOD2 (van de Wetering et al., 2008). These results suggest a link between apoptosis, oxidative stress and chromosome instability. The responses to metabolic perturbations in either B cell or T cell lymphomas were not known. In this work, we asked the following questions: 1) Will thymic tumor cells derived from Bax overexpressing mice versus Bcl-2 overexpressing mice differ in their histological and growth rate characteristics, frequency of p53 mutations, and sensitivity to metabolic perturbations? 2) Are Bcl-2 family proteins involved in regulating the response to metabolic perturbations in lymphomas?

32 14 3) Can 2-deoxy-D-glucose sensitize lymphomas to killing and therefore be considered as a treatment strategy? 4) What is the mechanism of 2-deoxy-D-glucose killing in lymphomas in relation to the Bcl-2 family? 5) Can this mechanism be manipulated to increase toxicity in cancer rather than in normal cells?

33 15 Figure 1. The Bcl-2 family of apoptotic regulators. The Bcl-2 family consists of a large group of genes that regulate apoptosis. The family is broadly divided into two functionally opposing groups, the antiapoptotic (prosurvival) and proapoptotic proteins. The proapoptotic group is further divided into two subgroups, the multidomain subgroup (Bax, Bak, and Bok) and the BH3-only proteins (Bim, Bik, Hrk, Noxa, Bid, Bad, Puma, and Bmf). The relative locations of the Bcl-2 homology (BH) and transmembrane (TM) domains are indicated. The homo- and hetero- dimerization of the Bcl-2 family members occurs through the BH3 domain. Adapted and modified from (Lessene et al., 2008).

34 16 Antiapoptotic Proapoptotic BH4 BH3 BH1 BH2 TM Multidomain BH3 BH1 BH2 TM BH3-only BH3 TM BH3 Bcl-2, Bcl-X L, Bcl-W, Mcl-1, A1 Bax, Bak, Bok Bim, Bik, Hrk, Noxa Bid, Bad, Puma, Bmf

35 17 Figure 2. Models of the Bcl-2 family members interactions. (A). Models for direct activation showing sensitization/direct activation and derepression/direct activation modes. (A1). In sensitization/direct activation modes: A BH3-only protein sensitizer (S) inhibits the antiapoptotic member (AA). Upon stress in the cell, a BH3-only direct activator (DA) is induced and not inactivated, going on to activate the proapoptotic effector (E) as shown in A3, therefore mitochondrial outer membrane permeabilization (MOMP) follows. (A2). In derepression /direct activation modes: A BH3-only direct activator (DA) is originally sequestered by the antiapoptotic member (AA). Upon stress delivery, BH3-only derepressor (DR) is upregulated and competes for binding with the antiapoptotic member (AA). With the BH3-only direct activator s (DA) release, it goes on to activate the proapoptotic effector (E), and MOMP follows. (A3). In direct activation mode, upon stress a BH3-only direct activator (DA) activates the proapoptotic effector (E), therefore MOMP follows. (B). In the neutralization model, proapoptotic effector (E) is actively inhibited by antiapoptotic member (AA). Upon stress delivery, BH3-only derepressor (DR) is upregulated, displacing proapoptotic effector Bax promoting MOMP.

36 18 A. Direct activation model A1. BH3-only sensitization/direct activation mode Stress Balance AA S DA DA AA S Proceed to A3 A2. BH3-only derepression/direct activation mode Stress Balance AA DA DR DA AA DR Proceed to A3 A3.Direct activation mode Stress Balance E B. Neutralization model Balance AA E active Stress DA DR E active E active AA DR MOMP MOMP

37 Figure 3. Chemical structures of D-glucose, D-mannose, and 2-deoxy-D-glucose. 19

38 20 O OH H H H HO OH H OH H OH O OH OH H H HO OH H H H OH O OH H H H HO OH H H OH H D-glucose D-mannose 2-Deoxy-D-glucose (2DG) 2-Deoxy-D-mannose (2DM) O OH H H H HO OH H OH H OH O OH OH H H HO OH H H H OH O OH H H H HO OH H H OH H D-glucose D-mannose 2-Deoxy-D-glucose (2DG) 2-Deoxy-D-mannose (2DM)

39 21 CHAPTER II T CELL LYMPHOMA (TCL) CELL LINES DERIVED FROM EITHER BAX OR BCL-2 OVEREPXPRESSION IN MICE DIFFER IN THEIR SENSITIVITY TO 2- DEOXY-D-GLUCOSE Summary Despite their opposing functions related to regulation of apoptosis, both Bax and Bcl-2 expression promote the development of T cell lymphoma. We hypothesized that the pathway to oncogenesis would be distinct in these two models. To test this hypothesis, T cell lymphomas were derived from both the Bax (Bax TCLs) and Bcl-2 (Bcl-2 TCLs) models and examined for cell cycle rate, p53 status, sensitivity to 2-deoxy- D-glucose (2DG) and by histology. The examination revealed that histological and growth rate characteristics were similar in both groups. Bax TCLs failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2 TCLs had near complete cell cycle arrest following radiation and rarely had mutations in p53. Importantly, Bcl-2 TCLs were uniformly resistant to 2DG treatment while Bax TCLs were all sensitive to 2DG to varying degrees. Thus, genetic factors such as the Bcl-2 family may prove useful when searching for biomarkers to predict response to treatment with 2DG. Introduction Over the years, scientific evidence has demonstrated the importance of the Bcl-2 family members in regulating apoptosis, whereby the proapoptotic group induces and the antiapoptotic group inhibits cell death. Aside from these defined roles of the Bcl-2

40 22 members in apoptosis, the studies also registered paradoxical effects of Bcl-2 family members in oncogenesis (de La Coste et al., 1999; Hogarth and Hall, 1999; Knudson et al., 2001; Rosenwald et al., 2002). This work examines murine lymphoma cell lines derived from thymic Bax or Bcl-2 overexpression. Bcl-2 has been shown to promote lymphoma in humans and in mice (Bakhshi et al., 1985; Cleary and Sklar, 1985; McDonnell et al., 1989; McDonnell and Korsmeyer, 1991; Tsujimoto et al., 1985). Despite this pro-oncogenic function, Bcl-2 has been shown to inhibit the cell cycle (Marvel et al., 1994; Vaux et al., 1988). The effects of Bcl-2 on cell cycle are mediated, at least in part, by p27, a Cyclin Dependent Kinase inhibitor (Vairo et al., 2000). To determine whether the effects of Bcl-2 on cell cycle inhibited the ability of Bcl-2 to promote lymphoma, Bcl-2 T cell transgenic mice were crossed to p27 deficient mice and examined for tumor formation. Thymic tumor formation was rapidly accelerated in these mice (Cheng et al., 2008). For the studies described here, cells isolated from thymic tumors in Bcl-2 p27-/- mice were used to generate Bcl-2 T cell lymphomas (Bcl-2 TCLs) to further characterize lymphomas caused by Bcl-2 overexpression. Despite its proapoptotic function, increased Bax expression accelerated lymphoma development in transgenic mice (Luke et al., 2003). The cell lines obtained from thymic tumors in Lck-Bax38/1 transgenic mice (T-cell restricted Bax overexpression) were used to generate Bax TCLs. Bax overexpression produced thymic tumors that are histologically very similar to tumors in Bcl-2 p27-/- mice. Given that Bax and Bcl-2 have opposing effects on cell viability, we hypothesized that the tumor cell lines derived from these two groups of mice would be phenotypically distinct. Consistent

41 23 with this hypothesis, Bax overexpression has been associated with chromosome instability and increased cell cycle progression (Knudson et al., 2001; Luke et al., 2003) while Bcl-2 overexpression attenuates aneuploidy (van de Wetering et al., 2007) and inhibits cell cycle progression (Belanger et al., 2005; Huang et al., 1997). It is also known that p53 deficiency cooperates with Bax expression to promote oncogenesis while Bcl-2 expression inhibits the inactivation of p53 during lymphomagenesis (Knudson et al., 2001; Schmitt et al., 2002). For the studies described here, cells isolated from thymic tumors in Lck-Bax38/1 mice were used to generate Bax T cell lymphoma (Bax TCL) cell lines. For the reasons described above, Bax TCLs were hypothesized to be distinct from Bcl-2 TCLs in their cell cycle rate and p53 status. Materials and Methods Cell Culture Thymic tumor cells (T Cell Lymphomas, TCLs) were obtained from five Lck- Bax38/1 mice and five p27 -/- Lck-Bcl-2 transgenic mice. Cells were harvested directly from thymic tumors of the transgenic mice and immediately minced into a single cell suspension between two frosted glass slides. The erythrocytes were removed by a fiveminute incubation at room temperature in hypotonic lysis buffer (10 mm Tris, 0.83% NH 4 Cl, ph 7.2). The cells were then pelleted and resuspended in complete medium [Advanced RPMI 1640 (Gibco) supplemented with 10% FBS, 2mM L-glutamine, 100 U/mL penicillin, 100 mg/l streptomycin, and 250 µg/l Fungizone Amphotericin (Invitrogen)]. Cells were maintained at 37 C in a humidified atmosphere of 5% CO 2 and 95% air. Thymic tumor cells grew in suspension. After cell growth was established, cells were frozen in liquid nitrogen for long term storage. Cell culture experiments were

42 24 restricted to the first four months of in vitro growth to avoid genomic drift. Doubling time (DT) of growing cells was calculated by the formula: DT = T/ logbase 2 (F/I), where T = time; F = Final cell count; I = Initial count. Note: Bax TCL2 stopped growing as the project evolved and was not available for monitoring cell growth, examining cell cycle arrest, or sequencing. Viability assay using Guava ViaCount reagent Cell viability and cell count were determined utilizing the flow cytometric Guava ViaCount assay that distinguishes between viable and non-viable cells based on the differential permeability of DNA-binding dyes in the ViaCount Reagent (Millipore) as previously described (Brown et al., 2007). Western Analysis Cell pellets were lysed in RIPA buffer (1% nonyl phenoxypolyethoxylethanol (NP40), 0.5% deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS) in phosphate buffered saline (PBS) containing Complete Protease Inhibitor Cocktail), incubated on ice for 30 min, pelleted at 13,000 rpm at 4 C for 30 min. The protein concentrations were determined by DC Bradford protein assay. Samples were brought to equal concentrations, mixed with the loading buffer containing 2-mercaptoethanol, boiled for 5 min, loaded and separated on a gradient (4-20%) Invitrogen Tris Glycine gel and then transferred to a nitrocellulose membrane. The membrane was blocked for 1 h in a 5% milk/pbs Tween solution and incubated overnight at 4 C with the primary antibody as previously described (van de Wetering et al., 2008). The membrane was washed three times over 15 min in PBS Tween and then incubated for 1 h at room temperature with secondary antibodies. The membrane was again washed three times over 15 min in PBS

43 25 Tween and then analyzed with ECL or ECL+ kit (Perkin Elmer). The following antibodies were used: Bcl-2 (Pharmingen, 15131S); p53 (Cell Signaling, 2524); β actin (Sigma, A4700); and GAPDH (AbCam Inc, ab9484), secondary Goat anti-rabbit (Caltag Laboratories), secondary Goat anti-mouse (Santa Cruz Biotechnology). Protein quantitation was assessed by the Bioimaging Systems, Ultra-Violet Products (UVP) and normalized to either actin of GAPDH. Ionizing Radiation Cells were resuspended in Adv.RPMI medium at equal concentrations, placed in 15 ml tubes, placed in the Gammacell 3000 chamber and irradiated for the indicated doses ( Rad) using a 137 Cesium γ-irradiation source (Gammacell 3000 Elan, Central Dose Rate 500 Rad/min, MDS Nordion). Cell Cycle Analysis by Propidium Iodide (PI) DNA content and BrdU uptake assays For PI-based cell cycle content analysis, cells were irradiated with 400 Rad and incubated 24 h. Cell cycle analysis was conducted by analyzing propidium iodidestained nuclei on a flow cytometer equipped for doublet discrimination (FACScan or FACSCalibur from Becton Dickinson). In brief, million cells were pelleted and resuspended in 0.5 ml of Krishan reagent prior to analysis by flow cytometry (Krishan, 1990). Doublet events were gated out based on FL-2 area versus FL-2 width. Cellquest software (Becton Dickinson) was used for acquisition, FlowJo software (Tree Star Inc.) was used for analysis. Cells in S phase (%) were estimated by Watson Pragmatic algorithm that is part of the FlowJo software.

44 26 For BrdU uptake experiments, cells were irradiated with 500 Rad, and after 24h exposed to 10 µm BrdU for 30 min in culture medium. Following BrdU exposure, cells were pelleted and fixed with ice-cold 70% ethanol and stored at 20 C prior to staining with anti-brdu Ab (Beckton Dickinson, ) and PI. Viable/nondoublet cells were analyzed based on FL-2 area versus FL-2 width dot plots. Cellquest software (Becton Dickinson) was used for acquisition, FlowJo software (Tree Star Inc.) was used for analysis. Reverse Transcriptase (RT)-PCR and sequencing The extraction of total cellular RNA from Bax and Bcl-2 TCLs was prepared using TRIzol reagent (Invitrogen Corp.), according to manufacturer s directions. The isolated RNA was quantitated spectrophotometrically by measuring the A260/A280 ratio. RNA (1 µg) was reverse transcribed to cdna using iscript Reverse Transcriptase kit (BioRad) (25 C 5 min x 42 C 30 min x 85 C 5 min x 4 C Hold). The reverse transcribed cdna was subjected to PCR amplification using primers for mouse p53 (sense, 5'-ATG- ACT-GCC-ATG-GAG-GAG-TCA-CAG-TCG-GAT-3'; antisense, 5'-CAG-TCT-GAG- TCA-GGC-CCC-ACT-3'). After an initial denaturation for 1 min at 94 C, cdna was amplified in a final volume 25 µl with 1.25U of TaqDNA polymerase using the manufacturer s buffer and 1.4 mm MgCl 2. Amplification of 1 µl of cdna was subjected to PCR for 30 cycles (94 C 1 min x 56 C 1 min x 72 C 1 min) finalized with annealing cycle (72 C 5 min x 4 C Hold). PCR products were run on 1% agarose gels containing ethidium bromide. Bands were first visualized with ethidium bromide and UV translumination. Second, band were then cut out from the gel and isolated using a

45 27 QIAquick Gel Extraction Kit (Qiagen). Samples were diluted in water and submitted for sequencing to the DNA Core Laboratory at the University of Iowa. Results Histological and growth rate characteristics are similar in thymic tumors derived from Bax or Bcl-2 overexpression To determine whether thymic tumors derived from mice with either Bax or Bcl-2 overexpression differed histologically, tumor tissue samples were examined by hematoxylin and eosin (H&E) (Fig. 4). No differences were observed among the two groups of thymic lymphoma tissues (N=4 from each group). To determine if there was a difference in growth characteristics between the two groups, the doubling time was measured during exponential cell growth (Table 1). Among four Bax TCLs, doubling time varied between 10 and 12 hours averaging at 11.0 hours. The doubling time for the four Bcl-2 TCLs was nearly identical ranging from 10 to 13 hours with an average of 11.2 hours. Overall, the rates of growth were comparable within each group as well as between the two groups. Thus, histologically and by growth rates, no significant differences were observed in thymic tumors that were a result of either Bax or Bcl-2 overexpression. Bax TCLs and Bcl-2 TCLs differ in their sensitivity to ionizing radiation We next determined how lymphomas derived from Bax or Bcl-2 overexpression respond to stress in the form of ionizing radiation (IR). Four Bax TCLs (1, 3 5) and five Bcl-2 TCLs (1 5) were irradiated (400 Rad) and monitored for cell viability and cell number for four days (Fig. 5A). All four Bax TCLs showed an initial drop in the