DOUBLE-STRAND BREAK REPAIR PATHWAYS IN DNA STRUCTURE-INDUCED GENETIC INSTABILITY

Genetic instability in mammalian cells can occur by many different mechanisms. In the absence of exogenous sources of DNA damage, the DNA structure itself has been implicated in genetic instability. When the canonical B-DNA helix is naturally altered to form a non-canonical DNA structure such as a Z-DNA or H-DNA, this can lead to genetic instability in the form of DNA double-strand breaks (DSBs) (1, 2). Our laboratory found that the stability of these non-B DNA structures was different in mammals versus Escherichia coli (E.coli) bacteria (1, 2). One explanation for the difference between these species may be a result of how DSBs are repaired within each species. Non-homologous end-joining (NHEJ) is primed to repair DSBs in mammalian cells, while bacteria that lack NHEJ (such as E.coli), utilize homologous recombination (HR) to repair DSBs. To investigate the role of the error-prone NHEJ repair pathway in DNA structure-induced genetic instability, E.coli cells were modified to express genes to allow for a functional NHEJ system under different HR backgrounds. The Mycobacterium tuberculosis NHEJ sufficient system is composed of Ku and Ligase D (LigD) (3). These inducible NHEJ components were expressed individually and together in E.coli cells, with or without functional HR (RecA/RecB), and the Z-DNA and H-DNA-induced mutations were characterized. The Z-DNA structure gave rise to higher mutation frequencies compared to the controls, regardless of the DSB repair pathway(s) available; however, the type of mutants produced after repair was greatly dictated on the available DSB repair system, indicated by the shift from 2% large-scale deletions in the total mutant population to 24% large-scale deletions when NHEJ was present (4). This suggests that NHEJ has a role in the large deletions induced by Z-DNA-forming sequences. H-DNA structure, however, did not exhibit an increase in mutagenesis in the newly engineered E.coli environment, suggesting the involvement of other factors in regulating H-DNA formation/stability in bacterial cells. Accurate repair by established DNA DSB repair pathways is essential to maintain the stability of eukaryotic and prokaryotic genomes and our results suggest that an error-prone NHEJ pathway was involved in non-B DNA structure-induced mutagenesis in both prokaryotes and eukaryotes.

14-3-3SIGMA NEGATIVELY REGULATES THE STABILITY AND SUBCELLULAR LOCALIZATION OF COP1

Mammalian constitutive photomorphogenic 1 (COP1), a p53 E3 ubiquitin ligase, is a key negative regulator for p53. DNA damage leads to the translocation of COP1 to the cytoplasm, but the underlying mechanism remains unknown. We discovered that 14-3-3σ controlled COP1 subcellular localization and protein stability. Investigation of the underlying mechanism suggested that, upon DNA damage, 14-3-3σ bound to phosphorylated COP1 at S387, resulting in COP1 translocation to the cytoplasm and cytoplasmic COP1 ubiquitination and proteasomal degradation. 14-3-3σ targeted COP1 for degradation to prevent COP1-mediated p53 degradation, p53 ubiquitination, and p53 transcription repression. COP1 expression promoted cell proliferation, cell transformation, and tumor progression, attesting to its role in cancer promotion. 14-3-3σ negatively regulated COP1 function and prevented tumor growth in cancer xenografts. COP1 protein levels were inversely correlated with 14-3-3σ protein levels in human breast and pancreatic cancer specimens. Together, these results define a novel, detailed mechanism for the posttranslational regulation of COP1 upon DNA damage and provide a mechanistic explanation of the correlation of COP1 overexpression with 14-3-3σ downregulation during tumorigenesis.

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

GCN5 maintains genome stability during development

The histone acetyltransferase, GCN5, is essential for survival of mice during embryogenesis. GCN5 null embryos die early during development due to increased apoptosis. We have demonstrated that the increased apoptosis in associated with increased p53 protein levels. Loss of p53 rescues the embryonic apoptosis in the GCN5 null embryos. These results raised the question of what molecular trigger leads to p53 stabilization and cell death in the absence of GCN5. p53 is generally referred to as the gatekeeper of the cell, monitoring cellular responses to DNA damage, genotoxic stress, and other unfavorable conditions in the cell. Therefore, we examined individual cells in wild type and mutant embryos for gross chromosomal aberrations that might trigger a genome integrity checkpoint. Karyotype analysis indicates that approximately 30% of the cells in an E8.5 GCN5 null embryo display chromosomal aberrations, predominantly chromosomal end adhesions and associations. In wild type E8.5 embryos, only 6% of the cells have chromosomal aberrations. Recent data using telomeric FISH demonstrates that cells from GCN5 null embryos have a decreased telomeric signal. Telomere maintenance is essential for maintaining genome integrity. Telomeric defects are associated with loss of chromosomes and chromosomal rearrangements that can lead to detrimental gene fusions involved in many types of cancers. Little is known about the chromatin structures present near the telomeric ends, or whether any of the telomere-associated proteins are subject to post-translational modification such as acetylation. Our results are the first data to demonstrate the involvement of a histone acetyltransferase, GCN5, in maintaining genome integrity through telomere maintenance and/or capping. ^

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

A systematic analysis of INO80 in DNA replication

ATP-dependent chromatin remodeling has been shown to be critical for transcription and DNA repair. However, the involvement of ATP-dependent chromatin remodeling in DNA replication remains poorly defined. Interestingly, we found that the INO80 chromatin-remodeling complex is directly involved in the DNA damage tolerance pathways activated during DNA replication. DNA damage tolerance is important for genomic stability and is controlled by formation of either mono-ubiquitinated or multi-ubiquitinated PCNA, which respectively induce error prone or error-free replication bypass of the lesions. In addition, homologous recombination (HR) mediated by the Rad51 pathway is also involved in the DNA damage tolerance pathways. ^ We found that INO80 is specifically recruited to replication origins during S phase in a genome-wide fashion. In addition, DNA combing analysis shows INO80 is required for the resumption of replication at stalled forks induced by methyl methane-sulfonate (MMS). Mechanistically, we find that INO80 is required for PCNA ubiquitination as well as for Rad51 mediated processing of replication forks after MMS treatment. Furthermore, chromatin immunoprecipitation at specific ARSs indicates INO80 is necessary for Rad18 and Rad51 recruitment to replication forks after MMS treatment. Moreover, 2D gel analysis shows INO80 is necessary to process Rad51 mediated intermediates at impeded replication forks. ^ In conclusion, our findings establish a novel role of a chromatin-remodeling complex in DNA damage tolerance pathways and suggest that chromatin remodeling is fundamentally important to ensure faithful replication of DNA and genome stability in eukaryotes. ^

Understanding the roles of Non-homologous end joining and p53 after DNA damage

The inability to maintain genomic stability and control proliferation are hallmarks of many cancers, which become exacerbated in the presence of unrepaired DNA damage. Such genotoxic stresses trigger the p53 tumor suppressor network to activate transient cell cycle arrest allowing for DNA repair; if the damage is excessive or irreparable, apoptosis or cellular senescence is triggered. One of the major DNA repair pathway that mends DNA double strand breaks is non-homologous end joining (NHEJ). Abrogating the NHEJ pathway leads to an accumulation of DNA damage in the lymphoid system that triggers p53-mediated apoptosis; complete deletion of p53 in this system leads to aggressive lymphomagenesis. Therefore, to study the effect of p53-dependent cell cycle arrest, we utilized a hypomorphic, separation-of-function mutant, p53p/p, which completely abrogates apoptosis yet retains partial cell cycle arrest ability. We crossed DNA ligase IV deficiency, a downstream ligase crucial in mending breaks during NHEJ, into the p53p/p background (Lig4-/-p53p/p). The accumulation of DNA damage activated the p53/p21 axis to trigger cellular senescence in developing lymphoid cells, which absolutely suppressed tumorigenesis. Interestingly, these mice progressively succumb to severe diabetes. Mechanistic analysis revealed that spontaneous DNA damage accumulated in the pancreatic b-cells, a unique subset of endocrine cells solely responsible for insulin production to regulate glucose homeostasis. The genesis of adult b-cells predominantly occurs through self-replication, therefore modulating cellular proliferation is an essential component for renewal. The progressive accumulation of DNA damage, caused by Lig4-/-, activated p53/p21-dependent cellular senescence in mutant pancreatic b-cells that lead to islet involution. Insulin levels subsequently decreased, deregulating glucose homeostasis driving overt diabetes. Our Lig4-/-p53p/p model aptly depicts the dichotomous role of cellular senescence—in the lymphoid system prevents tumorigenesis yet in the endocrine system leads to the decrease of insulin-producing cells causing diabetes. To further delineate the function of NHEJ in pancreatic b-cells, we analyzed mice deficient in another component of the NHEJ pathway, Ku70. Although most notable for its role in DNA damage recognition and repair within the NHEJ pathway, Ku70 has NHEJ-independent functions in telomere maintenance, apoptosis, and transcriptional regulation/repression. To our surprise, Ku70-/-p53p/p mutant mice displayed a stark increase in b-cell proliferation, resulting in islet expansion, heightened insulin levels and hypoglycemia. Augmented b-cell proliferation was accompanied with the stabilization of the canonical Wnt pathway, responsible for this phenotype. Interestingly, the progressive onset of cellular senescence prevented islet tumorigenesis. This study highlights Ku70 as an important modulator in not only maintaining genomic stability through NHEJ-dependent functions, but also reveals a novel NHEJ-independent function through regulation of pancreatic b-cell proliferation. Taken in aggregate, these studies underscore the importance for NHEJ to maintain genomic stability in b-cells as well as introduces a novel regulator for pancreatic b-cell proliferation.

FANCM AND FAAP24 MAINTAIN GENOMIC STABILITY THROUGH COOPERATIVE AND UNIQUE FUNCTIONS

Fanconi anemia (FA) is a rare recessive genetic disease with an array of clinical manifestations including multiple congenital abnormalities, progressive bone marrow failure and profound cancer susceptibility. A hallmark of cells derived from FA patients is hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC) and cisplatin, suggesting that FA- and FA-associated proteins play important roles in protecting cells from DNA interstrand crosslink (ICL) damage. Two genes involved in the FA pathway, FANCM and FAAP24, are of particular interest because they contain DNA interacting domains. However, there are no definitive patient mutations for these two genes, and the resulting lack of human genetic model system renders their functional studies difficult. In this study, I established isogenic human FANCM- and FAAP24-null mutants through homologous replacement-mediated gene targeting in HCT-116 cells, and systematically investigated the functions of FANCM and FAAP24 inchromosome stability, FA pathway activation, DNA damage checkpoint signaling, and ICL repair. I found that the FANCM-/-/FAAP24-/- double mutant was much more sensitive to DNA crosslinking agents than FANCM-/- and FAAP24-/- single mutants, suggesting that FANCM and FAAP24 possess epistatic as well as unique functions in response to ICL damage. I demonstrated that FANCM and FAAP24 coordinately support the activation of FA pathway by promoting chromatin localization of FA core complex and FANCD2 monoubiqutination. They also cooperatively function to suppress sister chromatid exchange and radial chromosome formation, likely by limiting crossovers in recombination repair. In addition, I defined novel non-overlapping functions of FANCM and FAAP24 in response to ICL damage. FAAP24 plays a major role in activating ICL-induced ATR-dependent checkpoint, which is independent of its interaction with FANCM. On the other hand, FANCM promotes recombination-independent ICL repair independently of FAAP24. Mechanistically, FANCM facilitates recruitment of nucleotide excision repair machinery and lesion bypass factors to ICL damage sites through its translocase activity. Collectively, my studies provide mechanistic insights into how genome integrity is both coordinately and independently protected by FANCM and FAAP24.

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

Endogenous nitric oxide regulates p53 signaling and DNA damage-induced apoptosis in melanoma cells

Nitric oxide is involved in a multitude of processes including regulation of vascular tone, neurotransmission, immunity, and cancer. Evidence suggests that nitric oxide exhibits anti-apoptotic activity in melanoma cells. Our laboratory showed that tumor expression of inducible nitric oxide synthase correlated strongly with poor survival in stage III and IV melanoma patients, suggesting an antagonistic role for nitric oxide in melanoma response to therapy. Therefore, the hypothesis that endogenously produced nitric oxide antagonizes chemotherapy-induced apoptosis was formed. Using cisplatin as a model for DNA damage in melanoma cell lines, the capacity of nitric oxide to regulate cell growth and apoptotic responses to cisplatin treatment was examined. The depletion of endogenously generated nitric oxide resulted in changes in cell cycle regulation and enhanced cisplatin-induced apoptosis in melanoma cells. Since nitric oxide was shown to be involved in the regulation of p53 stability, conformation and DNA binding activity, whether signaling through wild-type p53 in melanoma cells is regulated by nitric oxide was tested. Cisplatin-induced p53 accumulation and p21Waf1/Cip1/Sdi1 expression in nitric oxide-depleted melanoma cells were found to be strongly suppressed. When p53 binding to the p21Waf1/Cip1/Sdi1 promoter was examined, it was found that nitric oxide depletion significantly reduced the cisplatin-induced formation of p53-DNA complexes. These results suggest that nitric oxide is required for activation of wild-type p53 after DNA damage in melanoma cells. Finally, whether signaling through p53 controls melanoma response to DNA damage was examined. Transfection of a plasmid containing a dominant negative form of mutated p53 inhibited p21 Waf1/Cip1/Sdi1 expression and concomitantly enhanced apoptosis after cisplatin treatment. These data suggest that the induction of wild-type p53 protects melanoma cells against DNA damage via the up-regulation of p21 Waf1/Cip1/Sdi1. Together, these data strongly support the model that endogenous nitric oxide is required for p53 activation and p21Waf1/Cip1/Sdi1 expression after DNA damage, which can enhance melanoma resistance to therapy. Thus, in context of melanoma cells with wild-type p53 , low levels of endogenous constitutively-produced nitric oxide appear to facilitate the activation of p53 in response to DNA damage, thereby allowing for cell cycle arrest via p21Waf1/Cip1/Sdi1 induction, adequate DNA repair, and ultimately enhanced resistance to apoptosis. ^