Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2′-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.     

Synthesis of Mitomycin C and Decarbamoylmitomycin C N2 deoxyguanosine-adducts

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) – a derivative of MC lacking the carbamate on C10 – are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine–mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.     

Solution structure of a guanine-N7-linked complex of the mitomycin C metabolite 2,7-diaminomitosene and DNA. Basis of sequence selectivity

2,7-Diaminomitosene (2,7-DAM), the major metabolite of the antitumor antibiotic mitomycin C, forms DNA adducts in tumor cells. 2,7-DAM was reacted with the deoxyoligonucleotide d(GTGGTATACCAC) under reductive alkylation conditions. The resulting DNA adduct was characterized as d(G-T-G-[M]G-T-A-T-A-C-C-A-C) (5), where [M]G stands for a covalently modified guanine, linked at its N7-position to C10 of the mitosene. The adducted oligonucleotide complements with itself, retaining 2-fold symmetry in the 2:1 drug-duplex complex, and provides well-resolved NMR spectra, amenable for structure determination. Adduction at the N7-position of G4 ([M]G, 4) is characterized by a downfield shift of the G4(H8) proton and separate resonances for G4(NH(2)) protons. We assigned the exchangeable and nonexchangeable proton resonances of the mitosene and the deoxyoligonucleotide in adduct duplex 5 and identified intermolecular proton-proton NOEs necessary for structural characterization. Molecular dynamics computations guided by 126 intramolecular and 48 intermolecular distance restraints were performed to define the solution structure of the 2,7-DAM-DNA complex 5. A total of 12 structures were computed which exhibited pairwise rmsd values in the 0.54-1.42 A range. The 2,7-DAM molecule is anchored in the major groove of DNA by its C10 covalently linked to G4(N7) and is oriented 3' to the adducted guanine. The presence of 2,7-DAM in the major groove does not alter the overall B-DNA helical structure. Alignment in the major groove is a novel feature of the complexation of 2,7-DAM with DNA; other known major groove alkylators such as aflatoxin, possessing aromatic structural elements, form intercalated complexes. Thermal stability properties of the 2,7-DAM-DNA complex 5 were characteristic of nonintercalating guanine-N7 alkylating agents. Marked sequence selectivity of the alkylation by 2,7-DAM was observed, using a series of oligonucleotides incorporating variations of the 5'-TGGN sequence as substrates. The selectivity correlated with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA. The unusual, major groove-aligned structure of the adduct 5 may account for the low cytotoxicity of 2,7-DAM.     

Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.     

淋巴细胞凋亡与p53蛋白表达关系的研究

培养B95-8细胞,分离EB病毒,转染外周血和扁桃体淋巴细胞,建立永生化的LCLs和TLCL细胞株; 带有wt P₅₃基因的LCLs在DNA损伤剂——顺铂处理前未检出p53蛋白,经顺铂处理后,LCLs随作用时间延长细胞存活率明显下降、p53蛋白水平升高、DNA电泳显出梯状带;含mt P₅₃基因的淋巴瘤细胞在顺铂处理前可检出高浓度的p53蛋白,经顺铂处理后,细胞存活率与p53蛋白并无明显改变.这些结果表明：顺铂引起细胞DNA损伤、激活wt p53蛋白的表达、继而wt p53蛋白又促进了DNA损伤细胞凋亡.     

Evaluation of the effect of oxygen exposure on human liver microsomal metabolism of mitomycin C in the presence of glutathione using liquid chromatography-quadrupole time of flight mass spectrometry

This article describes a metabolite profiling method for evaluating the effect of oxygen exposure on human liver microsomal metabolism of mitomycin C (MC) in the presence of glutathione (GSH) and NADPH under hypoxic (100% helium), limitedly and fully aerobic, and hyperoxic (100% oxygen) conditions. MC and its metabolite(s) were characterized and the relative percentages of these components were estimated at different incubation times using liquid chromatography and quadrupole time-of-flight mass spectrometry. The MC metabolite profiles were confirmed using purified human cytochrome P450 reductase, acidic activation, and UV-Vis detection at 550 nm. In hypoxia, MC was exclusively metabolized into 2,7-diaminomitosene-10-glutathione-S-conjugate (2,7-DAM-10-SG) within 30 min, whereas approximately 5% of this conjugate, 16% of 2,7-diaminomitosene (2,7-DAM), and 77% of MC were observed under a fully aerobic condition at 90 min. Under limitedly aerobic conditions, the relative percentages of the two metabolites in incubations varied greatly depending on the volume ratio of air to liquid. In hyperoxia, 2% of 2,7-DAM-10-SG, 9% of 2,7-DAM, and 86% of MC were obtained at 90 min. The results indicate that oxygen strongly inhibits the in vitro metabolism of MC. These data suggest that GSH may serve a dual function in facilitating the formation of a leucoaziridinomitosene followed by electron rearrangement giving intermediate metabolite 2,7-DAM, and then trapping this intermediate giving rise to 2,7-DAM-10-SG. These findings provide direct evidence for understanding the fate of oxygen-sensitive metabolic deactivation of MC by GSH.     

ATR Activation Necessary But Not Sufficient for p53 Induction and Apoptosis in Hydroxyurea-Hypersensitive Myeloid Leukemia Cells

Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of the ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

Human urinary bladder epithelial cells lacking wild-type p53 function are deficient in the repair of 4-aminobiphenyl-DNA adducts in genomic DNA.

The effect of the tumor suppressor gene TP53 on repair of genomic DNA damage was examined in human urinary bladder transitional cell carcinoma (TCC) cell lines. Utilizing TCC10 containing wild-type p53 (wt-p53) as the parental line, an isogenic set of cell lines was derived by retroviral infection that expressed a transdominant mutant p53 (Arg --> His at codon 273, TDM273-TCC10), or the human papilloma virus 16-E6 oncoprotein (E6-TCC10). 32P-postlabeling analyses were performed on DNA from TCC cultures obtained after treatment with N-hydroxy-4-aminobiphenyl (N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP). The major adduct was identified as N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) with all three chemicals. The amount of adducts in urothelial DNA ranged between 0.1 and 20 per 10(6) nucleotides, N-OAc-AABP yielding the highest levels, followed by N-OH-ABP and N-OH-AABP. To determine, if the functional status of p53 affects the rate of repair of dG-C8-ABP in genomic DNA, TCC10 and the TDM273-TCC10 and E6-TCC10 isotypes were exposed to N-OH-AABP for 12h and the DNA damage was allowed to repair up to 24h. The adduct levels were quantified and compared between the TCC10 isotypes. The amounts of dG-C8-ABP that remained in genomic DNA from E6-TCC10 and TDM273-TCC10 were approximately two-fold higher, as compared to the parental TCC10. At the dose used for DNA repair studies, N-OH-AABP or N-OAc-AABP did not induce apoptosis in TCC10. However, N-OAc-AABP at high doses (>5 microM) induced apoptosis, as evidenced by DNA fragmentation analyses. Furthermore, N-OAc-AABP-mediated apoptosis was independent of the functional status of wt-p53, since both E6-TCC10 and the parental TCC10 exhibited DNA fragmentation following treatment. These results suggest that p53 might modulate the repair of DNA adducts generated from the human bladder carcinogen ABP in its target human uroepithelial cells. This implies that in p53 null cells the unrepaired DNA damage could cause accumulation of mutation, which might contribute to increased genomic instability and neoplastic progression.     

Bim directly antagonizes Bcl-xl in doxorubicin-induced prostate cancer cell apoptosis independently of p53

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

Taxol-induced mitotic block triggers rapid onset of a p53-independent apoptotic pathway.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.     

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.