The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

CISPLATIN NEPHROTOXICITY AND ITS EFFECTS ON GENTAMICIN PHARMACOKINETICS

Myelosuppression is a common side effect of anticancer agents such as cisplatin. This makes patients more susceptible to infections. Gentamicin is an aminoglycoside antibiotic that is very effective in the treatment of gram negative infections. Both these drugs are excreted by the kidney, and are also nephrotoxic. Thus, each may affect the disposition of the other. This project deals with the nature and duration of the effects of cisplatin on gentamicin pharmacokinetics in F-344 rats.^ The appropriate cisplatin dose was determined by comparing the nephrotoxicity of four intravenous doses--3, 4, 5, and 6 mg/kg. The 6 mg/kg dose gave the most consistent nephrotoxic effect, with peak plasma urea nitrogen and creatinine levels on the 7th day. Plasma and tissue gentamicin levels were compared between rats given gentamicin alone (30 mg/kg, intraperitoneally, twice a day for four days), and those given cisplatin (6 mg/kg, intraperitoneally) with the first gentamicin dose. Cisplatin caused a significant elevation of gentamicin levels in plasma, liver, and spleen. However, cisplatin given in three weekly doses of 2 mg/kg each, had no effect on plasma or tissue gentamicin levels.^ In order to determine the duration of cisplatin effects, a single dose of gentamicin (30 mg/kg, intravenously) was given to different groups of rats either alone, or on day 1, 4, 7, 15, or 29 following cisplatin (6 mg/kg, intravenously on day 1). Plasma samples were collected through a cannula placed on the external jugular vein at 0.5, 1, 2, 3, 4, 5, and 6 hours after gentamicin; the rats were sacrificed at 24 hours. Cisplatin caused a significant decrease in gentamicin excretion and an elevation of gentamicin levels in plasma, kidneys, liver, and spleen at all the time points that were tested, except with concomitant administration. Plasma urea nitrogen was elevated, and creatinine clearance decreased by the 4th day after cisplatin and these continued to be significantly different even on the 29th day after cisplatin.^ These results demonstrate that cisplatin nephrotoxicity reduced gentamicin excretion for at least a month in F-344 rats. This could increase the risk of toxicity from the second drug by elevating its levels in plasma and tissue. Thus, caution should be exercised when renally excreted drugs are given after cisplatin. ^

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

ATM Signaling to TSC2: Mechanisms and Implications for Cancer Therapy

Ataxia telangiectasia mutated (ATM) is a critical component of the cellular response to DNA damage, where it acts as a damage sensor, and signals to a large network of proteins which execute the important tasks involved in responding to the damage, namely inducing cell cycle checkpoints, inducing DNA repair, modulating transcriptional responses, and regulating cell death pathways if the damage cannot be repaired faithfully. We have now discovered that an additional novel component of this ATM-dependent damage response involves induction of autophagy in response to oxidative stress. In contrast to DNA damage-induced ATM activation however, oxidative stress induced ATM, occurs in the cytoplasm, and does not require nuclear-to-cytoplasmic shuttling of ATM. Using several cell culture systems including MCF7 breast carcinoma cells, SKOV3 ovarian cancer cells, and various lineages of mouse embryonic fibroblasts, we showed that once activated by reactive oxygen species (ROS), ATM signals to mTORC1 to induce autophagy via the LKB1-AMPK-TSC2 pathway. Targeting dysregulation of mTORC1 in Atm-deficient mice, which succumb to lymphomagenesis within 3-4 months of age with daily administration of rapamycin, could significantly extend survival and cause regression of tumors, suggesting that pharmacologically targeting this pathway has therapeutic implications in cancer. We also identified a second contrasting pathway for DNA damage-induced mTORC1 repression which does not require AMPK activation, but does require ATM and TSC2. Several potential mechanisms including mTOR localization and p53-mediated pathways were ruled out however we identified that TSC2 may be an additional cytoplasmic direct ATM substrate that is engaged in response to DNA damage specifically. Lastly, a study was performed to examine whether autophagy induced by ovarian cancer therapeutics (focusing on cisplatin, since paclitaxel does not induce autophagy in the SKOV3 cell line model we used) plays a role in resistance to therapy since autophagy can play both pro-survival mechanisms or be a mechanism of cell death. Using a genetic approach to knock-down Atg5 expression with shRNA in SKOV3 ovarian carcinoma cells, we compared the cytotoxicity of cisplatin in vector or Atg5 knock-down cells, and demonstrated that autophagy does not play any significant role in the response to cisplatin in this cell line.

Contribution of Ectodomain Mutations in Epidermal Growth Factor Receptor to Signaling in Glioblastoma Multiforme

CONTRIBUTION OF ECTODOMAIN MUTATIONS IN EPIDERMAL GROWTH FACTOR RECEPTOR TO SIGNALING IN GLIOBLASTOMA MULTIFORME Publication No._________ Marta Rojas, M.S. Supervisory Professor: Oliver Bögler, Ph.D. The Cancer Genome Atlas (TCGA) has conducted a comprehensive analysis of a large tumor cohort and has cataloged genetic alterations involving primary sequence variations and copy number aberrations of genes involved in key signaling pathways in glioblastoma (GBM). This dataset revealed missense ectodomain point mutations in epidermal growth factor receptor (EGFR), but the biological and clinical significance of these mutations is not well defined in the context of gliomas. In our study, we focused on understanding and defining the molecular mechanisms underlying the functions of EGFR ectodomain mutants. Using proteomic approaches to broadly analyze cell signaling, including antibody array and mass spectrometry-based methods, we found a differential spectrum of tyrosine phosphorylation across the EGFR ectodomain mutations that enabled us to stratify them into three main groups that correlate with either wild type EGFR (EGFR) or the long-studied mutant, EGFRvIII. Interestingly, one mutant shared characteristics of both groups suggesting a continuum of behaviors along which different mutants fall. Surprisingly, no substantial differences were seen in activation of classical downstream signaling pathways such as Akt and S6 pathways between these classes of mutants. Importantly, we demonstrated that ectodomain mutations lead to differential tumor growth capabilities in both in vitro (anchorage independent colony formation) and in vivo conditions (xenografts). Our data from the biological characterization allowed us to categorize the mutants into three main groups: the first group typified by EGFRvIII are mutations with a more aggressive phenotype including R108K and A289T; a second group characterized by a less aggressive phenotype exemplified by EGFR and the T263P mutation; and a third group which shared characteristics from both groups and is exemplified by the mutation A289D. In addition, we treated cells overexpressing the mutants with various agents employed in the clinic including temozolomide, cisplatin and tarceva. We found that cells overexpressing the mutants in general displayed resistance to the treatments. Our findings yield insights that help with the molecular characterization of these mutants. In addition, our results from the drug studies might be valuable in explaining differential responses to specific treatments in GBM patients.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

Molecular alterations in nucleotide excision repair genes in malignant glioma and their relevance to malignant progression and drug resistance

Recently, it has become apparent that DNA repair mechanisms are involved in the malignant progression and resistance to therapy of gliomas. Many investigators have shown that increased levels of O6-methyl guanine DNA alkyltransferase, a DNA monoalkyl adduct repair enzyme, are correlated with resistance of malignant glioma cell lines to nitrosourea-based chemotherapy. Three important DNA excision repair genes ERCC1 (excision repair cross complementation group 1), ERCC2 (excision repair cross complementation group 2), and ERCC6 (excision repair cross complementation group 6) have been studied in human tumors. Gene copy number variation of ERCC1 and ERCC2 has been observed in primary glioma tissues. A number of reports describing a relationship between ERCC1 gene alterations and resistance to anti-cancer drugs have been also described. The levels of ERCC1 gene expression, however, have not been correlated with drug resistance in gliomas. The expression of ERCC6 gene transcribes has been shown to vary with tissue types and to be highest in the brain. There have been no comprehensive studies so far, however, of ERCC6 gene expression and molecular alterations in malignant glioma. This project examined the ERCC1 expression levels and correlated them with cisplatin resistance in malignant glioma cell lines. We also examined the molecular alterations of ERCC6 gene in primary glioma tissues and cells and analyzed whether these alterations are related to tumor progression and chemotherapy resistance. Our results indicate the presence of mutations and/or deletions in exons II and V of the ERCC6 gene, and these alterations are more frequent in exon II. Furthermore, the mutations and/or deletions in exon II were shown to be associated with increased malignant grade of gliomas. The results on the Levels of ERCC1 gene transcripts showed that expression levels correlate with cisplatin resistance. The increase in ERCC1 mRNA induced by cisplatin could be down-regulated by cyclosporin A and herbimycin A. The results of this study are likely to provide useful information for clinical treatment of human gliomas. ^

Pharmacogenetics of the human glutathione S-transferase P1 gene in the metabolism and therapeutic efficacy of cis-diamminedichloroplatinum

The human GSTP1 gene has been shown, conclusively, to be polymorphic. The three main GSTP1 alleles, GSTP1*A, GSTP1*B, and GSTP1*C, encode proteins which differ in the 3-dimensional structure of their active sites and in their function in phase II metabolism of carcinogens, mutagens, and anticancer agents. Although, it is well established that GSTP1 is over expressed in many human tumors and that the levels of GSTP1 expression correlate directly with tumor resistance to chemotherapy and inversely with patient survival, the significance of the polymorphic GSTP1 gene locus on tumor response to chemotherapy remains unclear. The goal of this project was to define the role and significance of the polymorphic GSTP1 gene locus in GSTP1-based tumor drug resistance and as a determinant of patient response to chemotherapy. The hypothesis to be tested was that the polymorphic GSTP1 gene locus will confer to tumors a differential ability to metabolize cisplatin resulting in a GSTP1 genotype-based sensitivity to cisplatin. The study examined: (a) whether the different GSTP 1 alleles confer different levels of cellular protection against cisplatin-induced cytotoxicity, (b) whether the allelic GSTP1 proteins metabolize cisplatin with different efficiencies, and (c) whether the GSTP1 genotype is a determinant of tumor response to cisplatin therapy. The results demonstrate that the GSTP1 alleles differentially protect tumors against cisplatin-induced apoptosis and clonogenic cell kill in the rank order: GSTP1*C > GSTP1*B > GSTP1*A. The same rank order was observed for the kinetics of GSTP1-catalyzed cisplatin metabolism, both in cell-free and cellular systems, to the rate-limiting monoglutathionyl-platinum metabolite, which was characterized, for the first time, by mass spectral analysis. Finally, this study demonstrates that both GSTP1 genotype and the level of GSTP1 expression significantly contribute to tumor sensitivity to cisplatin treatment. Overall, the results of this project show that the polymorphic GSTP1 gene locus plays a significant role in tumor sensitivity to cisplatin treatment. Furthermore, these studies have contributed to the overall understanding of the significance of the polymorphic GSTP1 gene locus in tumor resistance to cancer chemotherapy and have provided the basis for further investigations into how this can be utilized to optimize and individualize cancer chemotherapy for cancer patients. ^

Roles of mismatch repair proteins in the recognition and processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) are among the most toxic type of damage to a cell. Many ICL-inducing agents are widely used as therapeutic agents, e.g. cisplatin, psoralen. A bettor understanding of the cellular mechanism that eliminates ICLs is important for the improvement of human health. However, ICL repair is still poorly understood in mammals. Using a triplex-directed site-specific ICL model, we studied the roles of mismatch repair (MMR) proteins in ICL repair in human cells. We are also interested in using psoralen-conjugated triplex-forming oligonucleotides (TFOs) to direct ICLs to a specific site in targeted DNA and in the mammalian genomes. ^ MSH2 protein is the common subunit of two MMR recognition complexes, and MutSα and MutSβ. We showed that MSH2 deficiency renders human cell hypersensitive to psoralen ICLs. MMR recognition complexes bind specifically to triplex-directed psoralen ICLs in vitro. Together with the fact that psoralen ICL-induced repair synthesis is dramatically decreased in MSH2 deficient cell extracts, we demonstrated that MSH2 function is critical for the recognition and processing of psoralen ICLs in human cells. Interestingly, lack of MSH2 does not reduce the level of psoralen ICL-induced mutagenesis in human cells, suggesting that MSH2 does not contribute to error-generating repair of psoralen ICLs, and therefore, may represent a novel error-free mechanism for repairing ICLs. We also studied the role of MLH1, anther key protein in MMR, in the processing of psoralen ICLs. MLH1-deficient human cells are more resistant to psoralen plus UVA treatment. Importantly, MLH1 function is not required for the mutagenic repair of psoralen ICLs, suggesting that it is not involved in the error-generating repair of this type of DNA damage in human cells. ^ These are the first data indicating mismatch repair proteins may participate in a relatively error-free mechanism for processing psoralen ICL in human cells. Enhancement of MMR protein function relative to nucleotide excision repair proteins may reduce the mutagenesis caused by DNA ICLs in humans. ^ In order to specifically target ICLs to mammalian genes, we identified novel TFO target sequences in mouse and human genomes. Using this information, many critical mammalian genes can now be targeted by TFOs.^

INACTIVATION OF MUTAGENIC DRUGS AND THEIR METABOLITES IN THE URINE OF PATIENTS ADMINISTERED ANTINEOPLASTIC THERAPY

Urines from patients administered mutagenic antineoplastic drugs were significantly mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urines were tested for mutagenicity in several different strains of Salmonella typhimurium that were uvr positive or negative (TA98, TA100, TA102, UTH8413, UTH8414). The urines were fractionated by high pressure liquid chromatography (HPLC) and the fractions assayed for mutagenicity in the strains in which the whole urine was mutagenic. Only fractions of urines containing the parent compound (cisplatin, doxorubicin, or mitomycin) were mutagenic; no other fraction showed significant mutagenicity. However, urine containing cyclophosphamide had two fractions that were mutagenic. One fraction, the fraction containing cyclophosphamide, required metabolic activation for mutagenicity. The other fraction did not require activation for mutagenicity.^ The chemical and mutagenic stability of these urines at room temperature was assayed over a 14 day period. The parent compound degraded within the first seven days, but the urines remained mutagenic. Cis-platinum was chemically stable in the urine; however, the urine decreased in mutagenicity. The decrease was probably the result of stable ligands binding to the platinum.^ Inactivation methods were developed to reduce the genotoxic hazard. Urine containing cisplatin was inactivated by complexing the cisplatin with diethyldithiocarbamate (DDTC). Oxidation with NaOCl of urines containing mitomycin and doxorubicin (sodium thiosulfate must be added to the doxorubicin urine) results in mutagenic inactivation. Inactivation of urine containing cyclophosphamide requires oxidation with alkaline potassium permaganate and trapping of active degradation products with sodium thiosulfate. Urines containing these drugs can be inactivated, but not always by the same method that inactivates the drug alone in solution. Therefore, in the future development of inactivation methods, both chemical and mutagenic assays are necessary to determine effectiveness. Methods of inactivation of mutagenic excreta developed in this study are both effective and practical. ^

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.

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. ^

Role of the glutathione S -transferase P1 (GSTP1) electrophile binding site (H-site) in protection from doxorubicin -mediated cytotoxicity

The hypothesis addressed in this project was that novel variants of naturally occurring human glutathione S-transferase P1 (GSTP1) can be created by random mutagenesis of the GSTP1 active site to yield polypeptides with increased enzymatic activity against electrophilic substrates. Specifically, the mutant proteins would metabolize and inactivate selected electrophiles more efficiently than wild-type GSTP1 and confer significant cytoprotection, as measured by reduced apoptosis and increased clonogenic survival. Glutathione S-transferase P1, a major electrophile metabolizing and detoxifying enzyme, is encoded by a polymorphic genetic locus. This locus contains nucleotide transitions in the region encoding the active site of the peptide that yields proteins with significant structural and functional differences. The method of Degenerate Oligonucleotide Mediated Random Mutagenesis (DOMRM) was used to generate cDNAs encoding unique GSTP1 polypeptides with mutations within electrophile binding site (H-site) while leaving the glutathione binding site unaffected. A prokaryotic expression library of the mutant GSTP1 polypeptides was created and screened for increased resistance to cisplatin. This screen resulted in the isolation of 96 clones representing 22 distinct mutant cDNA sequences. To investigate the effects of the changes in the H-site on the biological activity of GSTP1, the cDNA of wild-type GSTP1c and two of the identified mutants were stably transfected into human LNCaP-Pro5 prostate cancer cells that do not endogenously express GSTP1. Wild-type transfectants were resistant to doxorubicin-induced apoptosis and displayed increased clonogenic survival compared to vector controls. However, contrary to the hypothesis, in both assays the mutant transfectants were no more resistant to doxorubicin than the wild-type transfectants. To elucidate the mechanisms underlying GSTP1-mediated survival, an in-vitro assay was developed to determine whether active GSTP1 protein directly metabolizes doxorubicin by conjugation to reduced glutathione (GSH). Although GSH did promote the appearance of a unique doxorubicin conjugate, conjugate formation was not substantially increased by the addition of GSTP1 in a variety of reaction conditions. ^