Repair Of Adducts Research Articles

MDM2 sensitizes a human ovarian cancer cell line

Objectives The purpose of this study was to determine whether overexpression of MDM2 could sensitize the ovarian cancer cell line A2780. Methods The wild-type p53-expressing cell line A2780 was stably transfected with pCMV-MDM2 (A2780-MDM2) or pCMV (A2780-V) as control. MTT assay and clonogenic survival assay were used to measure the cisplatin sensitivity. FACS and host cell (CAT) reactivation assay were used to estimate the change of cell cycle and ability of repairing cisplatin-induced DNA damage. Results Parental A2780 and A2780-V had similar cisplatin sensitivities, whereas A2780-MDM2 was two- to threefold more sensitive to cisplatin. Repair of cisplatin-induced DNA damage was reduced in A2780 cells overexpressing MDM2, compared to A2780 cells in which wild-type p53 function was intact. After cisplatin treatment, A2780-MDM2 cells showed a pronounced S-phase arrest; however, A2780 cells with intact wild-type p53 arrested primarily in G2/M phase. Conclusions MDM2 overexpression can increase cisplatin cytotoxicity in A2780, with loss of G1/S checkpoint control and decreased cisplatin–DNA adduct repair. This suggests that ovarian cancers that overexpress MDM2 may be amenable to treatment with platinum compounds.

SU5416 sensitizes ovarian cancer cells to cisplatin through inhibition of nucleotide excision repair.

SU5416 is reported to be a selective inhibitor of vascular endothelial growth factor, and it has metwith limited success in the clinic. In the present study, we investigated whether SU5416 could augment cisplatin-induced cytotoxicity in human ovarian cancer cells. When used as a single agent, 2-h exposures to SU5416 were not harmful to the cells up to doses of 100 microM. For 48-h exposures, the SU5416 IC20 and IC50 were 17 and 34 microM, respectively. When used with cisplatin, the effect of SU5416 was sequence dependent. SU5416 given first was subadditive, whereas cisplatin given first was supraadditive. Cisplatin was given as a 1-h exposure. Augmented cisplatin cytotoxicity was seen with 2-h exposures to SU5416 at doses of 17-34 microM. This was associated with a decrease in cisplatin-DNA adduct repair, as measured by atomic absorbance spectrometry. Treatment of the ovarian carcinoma cells with SU5416 was also associated with a reduced expression of ERCC-1 protein and c-jun mRNA, as well as a decrease in c-Jun and JNK activities. We conclude that SU5416 can be used to augment cisplatin-induced cell killing at doses that are non-toxic. This effect may occur through direct or indirect reduction of the activity of AP-1 and DNA repair.

Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes.

We have shown previously that a wide range of mutagenic carcinogens are capable of inducing loss of heterozygosity (LOH) at the endogenous Aprt locus in mouse splenic lymphocytes. To investigate whether LOH might be caused by a single common mechanism, we set out to determine the extent of LOH by microsatellite analysis along (the Aprt gene containing) mouse chromosome 8. Aprt+/- hybrid B6C3F1 mice were treated with mutagens that induce different classes of DNA lesions, i.e. bulky DNA adducts, DNA methylation, DNA inter-strand crosslinks or DNA strand breaks. Aprt mutant frequencies (MF) in this C57Bl/6-C3H hybrid background were significantly reduced for mitomycin C (MMC) and methylmethanesulfonate (MMS) in comparison with MF in C57Bl/6 background, suggesting either enhanced repair or reduced formation of MMC- or MMS-induced mutagenic lesions in a hybrid B6C3F1 background. In contrast, Aprt MF after dimethylbenz[a]anthracene (DMBA), methylnitrosurea (MNU) and etoposide treatment were similar in both genetic backgrounds. Microsatellite analysis of Aprt mutant clones indicated a dominant role for mitotic recombination (MR) in generating spontaneous, DMBA- and etoposide-induced LOH at APRT: However, over 80% of the MMC-induced Aprt LOH mutants had lost heterozygosity for all markers tested, suggesting that either the crossover points were located close to the centromere or that these mutants arose by chromosome loss and duplication of the remaining chromosome 8. A substantial fraction (40%) of MNU-induced Aprt mutants had lost the wild-type Aprt allele, but had retained heterozygosity at all polymorphic markers tested at chromosome 8 indicating an important role for deletions in LOH formation by MNU. Patterns of MR differed quite dramatically for the various chemical mutagens tested, suggesting different mechanisms to be involved in inducing recombination between homologous chromosomes. In addition, non-random adduct formation and repair between chromosomal regions, i.e. heterochromatin versus euchromatin, may contribute to a non-random distribution of recombinational crossover points.

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts.

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.

DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin

The influence of DNA repair mechanisms on the interaction between gemcitabine and cisplatin was studied using a panel of Chinese hamster ovary (CHO) cell lines each deficient in one of the following repair pathways: base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ). NER and HR are known to be involved in platinum-DNA adduct repair. Single agent experiments demonstrated that each of the repair deficient cell lines had a similar sensitivity towards gemcitabine as the parental cell lines, whereas the NER- and HR-deficient lines showed increased sensitivity towards cisplatin. Furthermore, in the parental cell lines, the administration sequence cisplatin followed by gemcitabine was synergistic, whereas the reversed schedule showed additivity and simultaneous administration revealed antagonistic cytotoxicity. In the repair deficient cell lines, using this synergistic schedule of cisplatin followed by gemcitabine, loss of synergy was observed in the NER- and HR-deficient cell lines. However, the magnitude of the effect in the NER-deficient cells was small. The sensitivity to the combination of cisplatin and gemcitabine shown by the BER- and NHEJ-deficient cell lines did not differ significantly from that of the parental cell line. Cellular accumulation of platinum as well as the formation of GG- and AG-intrastrand adducts in the parental line and in the HR-deficient line were not affected by gemcitabine. In conclusion, our results indicate that BER, NER, HR, and NHEJ are most likely incapable of modulating the cytotoxicity of gemcitabine, and that HR is involved in the synergistic interaction between cisplatin and gemcitabine in our cell system.

Saccharomyces cerevisiae RAD5 Influences the Excision Repair of DNA Minor Groove Adducts

Nucleotide excision repair (NER) is the primary pathway for the removal of DNA adducts that distort the double helix. In the yeast Saccharomyces cerevisiae the RAD6 epistasis group defines a more poorly characterized set of DNA damage response pathways, believed to be distinct from NER. Here we show that the elimination of the DNA minor groove adducts formed by an important class of anticancer antibiotic (CC-1065 family) requires NER factors in S. cerevisiae. We also demonstrate that the elimination of this class of minor groove adduct from the active MFA2 gene depends upon functional Rad18 and Rad6. This is most clear for the repair of adducts on the transcribed strand, where an absolute requirement for Rad6 and Rad18 was seen. Further experiments revealed that a specific RAD6-RAD18-controlled subpathway, the RAD5 branch, mediates these events. Cells disrupted for rad5 are highly sensitive to this minor groove binding agent, and rad5 cells exhibit an in vivo adduct elimination defect indistinguishable from that seen in rad6 and rad18 cells as well as in NER-defective cells. Our results indicate that the RAD5 subpathway may interact with NER factors during the repair of certain DNA adducts.

ERCC2/XPD gene polymorphisms and cancer risk.

DNA repair of bulky adducts is essential for a normal life, as demonstrated by the existence of rare but dramatic diseases, such as xeroderma pigmentosum (XP), associating DNA repair deficiency and a high cancer proneness. It is plausible that small variations in the efficacy of repair in the normal population may facilitate cancer development in exposed individuals. In order to check this hypothesis, associations between single nucleotide polymorphisms (SNPs) in key genes and some frequent human cancers have been researched. Among the repair proteins, the XPD protein is interesting because it is a major player in the nucleotide excision repair pathway and is also involved in transcription initiation and in the control of the cell cycle and apoptosis. Several SNPs have been described in the ERCC2/XPD gene, but three in particular have been studied: the C-->A silent polymorphism (Arg156Arg) in exon 6, the G-->A polymorphism leading to Asp312Asn in exon 10 and the A-->C polymorphism leading to Lys751Gln in exon 23. We review here the epidemiological studies examining whether these polymorphisms are correlated with reduced DNA repair efficiency (analysed using different assays), their influence on the development of cutaneous carcinomas and smoking-related cancers and their possible interactions with environmental exposures.

Mechanism of nucleotide excision repair of DNA adducts formed by chemical carcinogens and antitumor drugs

Mechanism of nucleotide excision repair of DNA adducts formed by chemical carcinogens and antitumor drugs

Hypersensitivity of DNA polymerase β null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions

Monofunctional alkylating agents react with DNA by S N 1 or S N 2 mechanisms resulting in formation of a wide spectrum of cytotoxic base adducts. DNA polymerase β (β-pol) is required for efficient base excision repair of N-alkyl adducts, and we make use of the hypersensitivity of β-pol null mouse fibroblasts to investigate such alkylating agents with a view towards understanding the DNA lesions responsible for the cellular phenotype. The inability of O 6-benzylguanine to sensitize wild-type or β-pol null cells to S N 1-type methylating agents indicates that the observed hypersensitivity is not due to differential repair of cytotoxic O-alkyl adducts. Using a 3-methyladenine-specific agent and an inhibitor of such methylation, we find that inefficient repair of 3-methyladenine is not the reason for the hypersensitivity of β-pol null cells to methylating agents, and further that 3-methyladenine is not the adduct primarily responsible for methyl methanesulfonate (MMS)- and methyl nitrosourea-induced cytotoxicity in wild-type cells. Relating the expected spectrum of DNA adducts and the relative sensitivity of cells to monofunctional alkylating agents, we propose that the hypersensitivity of β-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5′-deoxyribose phosphate group, following removal of 7-alkylguanine from DNA. In support of this conclusion, β-pol null cells are also hypersensitive to the thymidine analog 5-hydroxymethyl-2′-deoxyuridine (hmdUrd). This agent is incorporated into cellular DNA and elicits cytotoxicity only when removed by glycosylase-initiated base excision repair. Consistent with the hypothesis that there is a common repair intermediate resulting in cytotoxicity following treatment with both types of agents, both MMS and hmdUrd-initiated cell death are preceded by a similar rapid concentration-dependent suppression of DNA synthesis and a later cell cycle arrest in G 0/G 1 and G 2M phases.

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer.

Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.

Interference by toxic metal ions with DNA repair processes and cell cycle control: molecular mechanisms.

Nickel, cadmium, cobalt, and arsenic compounds are well-known carcinogens to humans and experimental animals. Even though their DNA-damaging potentials are rather weak, they interfere with the nucleotide and base excision repair at low, noncytotoxic concentrations. For example, both water-soluble Ni(II) and particulate black NiO greatly reduced the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant. Furthermore, Ni(II), As(III), and Co(II) interfered with cell cycle progression and cell cycle control in response to ultraviolet C radiation. As potential molecular targets, interactions with so-called zinc finger proteins involved in DNA repair and/or DNA damage signaling were investigated. We observed an inactivation of the bacterial formamidopyrimidine-DNA glycosylase (Fpg), the mammalian xeroderma pigmentosum group A protein (XPA), and the poly(adenosine diphosphate-ribose)polymerase (PARP). Although all proteins were inhibited by Cd(II) and Cu(II), XPA and PARP but not Fpg were inhibited by Co(II) and Ni(II). As(III) deserves special attention, as it inactivated only PARP, but did so at very low concentrations starting from 10 nM. Because DNA is permanently damaged by endogenous and environmental factors, functioning processing of DNA lesions is an important prerequisite for maintaining genomic integrity; its inactivation by metal compounds may therefore constitute an important mechanism of metal-related carcinogenicity.

Recognition and repair of DNA-cisplatin adducts.

Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intra- and interstrand crosslinks. DNA-cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA-protein and protein-protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA-cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA-cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity.

Recognition and repair of DNA-cisplatin adducts.

Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intra- and interstrand crosslinks. DNA-cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA-protein and protein-protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA-cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA-cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity.

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin.

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.

Molecular aspects of resistance to antitumor platinum drugs.

The processes by which cells develop resistance to antitumor platinum drugs have been the subject of intense research because resistance is a major obstacle for the clinical use of this class of drugs. It is therefore of great interest to understand the molecular and biochemical mechanisms that underlie resistance to platinum drugs and their biological effects. There is a large body of experimental evidence suggesting that the antitumor activity of platinum complexes stems from their ability to form on DNA various types of covalent adducts. As a result, research on DNA modifications by these drugs and their cellular processing has predominated. The resistance of tumor cells to platinum drugs has been attributed to several processes and an increased repair of platinum-DNA adducts is considered a most significant event. The present review summarizes recent insights into the effects of sulfur-containing compounds on DNA modifications by antitumor platinum complexes and how these modifications are repaired including how this repair is associated with their recognition by cellular, damaged-DNA binding-proteins. It strongly supports the view that changes in the structure of platinum drugs, resulting in DNA binding mode fundamentally different from that of "classical" cisplatin, will alter resistance pathways of platinum drugs, and may also modulate their pharmacological properties.

Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21 waf1/cip1 and pRb

Wild-type p53 protein is known to regulate the global genomic repair (GGR), removing bulky chemical DNA adducts as well as cyclobutane pyrimidine dimers from the genome overall and from non-transcribed strands (NTS) in DNA. To investigate the role of cellular factor(s) relevant to p53 regulated DNA repair processes, we examined the repair kinetics of chemical carcinogen, anti-benzo[a]pyrene-diol epoxide (anti-BPDE), induced bulky DNA adducts in normal human mammary epithelial cells (HMECs) and HMEC transformed by human papillomavirus (HPV)-16E6 or -16E7 oncoproteins, which, respectively targets p53 or pRb proteins for degradation. The results show that the removal of anti-BPDE DNA adducts from the genome overall and NTS by GGR was significantly reduced in HPV-16E6 protein expressing cells as compared to that in normal and HPV-16E7 protein expressing cells, indicating the role of p53 and not pRb in nucleotide excision repair (NER). We further determined the potential effects of the p53-regulated p21 waf1/cip1 gene product in NER in human colon carcinoma, HCT116 cells expressing wild-type p53 but different p21 waf1/cip1 genotypes (p21+/+, p21+/−, p21−/−). The results donot show a discernible difference in the removal of anti-BPDE DNA adducts from the genome overall and the transcribed strand (TS) and NTS irrespective of the presence or absence of p21 waf1/cip1 expression. Based on these results, we suggest that: (i) the wild-type p53 function but not p21 waf1/cip1 expression is necessary for GGR of chemical induced bulky DNA adducts; (ii) the Rb gene product does not play a significant role in NER; and (iii) the modulation of NER by p53 may be independent of its function in the regulation of cell cycle arrest upon chemically induced DNA damage.

The XPD variant alleles are associated with increased aromatic DNA adduct level and lung cancer risk

The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is involved in the nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. In order to study the functional impact of the common polymorphisms in XPD exon 10 (G > A, Asp312Asn) and exon 23 (A > C, Lys751Gln), we have genotyped 185 Swedish lung cancer cases (97 smokers and 88 never-smokers) and 162 matched population controls (83 smokers and 79 never-smokers). Presence of one or two variant alleles was associated with increased risk for lung cancer among never-smokers only, in particular younger (<70 years) never-smokers [odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.1-6.5 for exon 10; OR = 3.2, 95% CI = 1.3-8.0 for exon 23, adjusted for age, gender and environmental tobacco smoke]. Aromatic DNA adduct level (AL) in peripheral lymphocytes was found to be similar between cases and controls, but significantly increased by current or recent smoking. Overall, there was a significant trend for increasing AL with increasing number of variant alleles in exon 10 (P = 0.02) or in exon 23 (P = 0.001). In addition, subjects with the combined exon 10 AA and exon 23 CC genotype showed a significantly higher AL compared with all those with any of the other genotypes (P = 0.02). We conclude that the XPD variant alleles may be associated with reduced repair of aromatic DNA adducts in general and increased lung cancer risk among never-smokers.

Developmental chemotherapy in advanced ovarian cancer: Incorporation of newer cytotoxic agents in a phase III randomized trial of the Gynecologic Oncology Group (GOG-0182)

Despite improvements in median and overall survival using a combination of platinum and paclitaxel, long-term survival rates for patients with advanced epithelial ovarian carcinoma (EOC) remain disappointing, and the development of more effective primary therapy remains a priority. In particular, several interesting chemotherapy agents have demonstrated activity individually in patients with recurrent EOC. Among these are gemcitabine, topotecan, liposomal doxorubicin, and prolonged oral etoposide. Preclinical models have suggested an advantage for combinations of these agents with platinum, which has been attributed to inhibition of DNA synthetic pathways involved in the repair of platinum-DNA adducts. However, efforts to develop multidrug combinations with platinum and paclitaxel have encountered substantial bone marrow toxicity, prompting exploration of alternative schedules and sequences of drug administration. In this regard, the Gynecologic Oncology Group (GOG) and other organizations have conducted a series of phase I pilot studies in previously untreated patients to define combinations that are suitable for group-wide phase III trials. With international collaboration, GOG has launched a five-arm trial (GOG-0182) that will compare these combinations against carboplatin-paclitaxel. The selection of candidate regimens for this trial illustrates the challenges of drug development in EOC. Semin Oncol 29 (suppl 1):20-31. Copyright © 2002 by W.B. Saunders Company.

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 12 h and the DNA damage was allowed to repair up to 24 h. 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 μM) 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.

Combination of oxaliplatin and irinotecan on human colon cancer cell lines: activity in vitro and in vivo.

The in vitro and in vivo combination of oxaliplatin and irinotecan was investigated in a panel of four human colon cancer cell lines and their counterpart xenografts. In vitro and in vivo experiments demonstrated a synergistic or additive interaction in three cell lines (HCT-116, HCT-8 and HT-29) and an antagonism in SW-620 cells. Since there were clearly opposite interactions depending on the cell line, we further investigated cellular determinants possibly involved in the interaction between the two drugs in HCT-8 and SW-620 cells. Irinotecan slowed down the early platinum-DNA adducts repair (1 h after oxaliplatin exposure) in the presence of irinotecan only in HCT-8 cells (p=0.03, n=3). Moreover, a decrease of the expression of two proteins of the nucleotide excision repair (NER) system, ERCC1 and XPA, was observed. None of these effects was seen in SW-620 cells. Irinotecan induced apoptosis with an increase of poly(ADP-ribose) polymerase (PARP) cleavage in SW-620 cells (60 versus 7% basal level). Pretreatment of these cells with oxaliplatin abolished the increase in PARP cleavage induced by irinotecan (29%). In HCT-8 cells, a very little PARP cleavage was observed whatever the drug treatment. The persistence of platinum-DNA adducts in the presence of irinotecan could be due to a direct impact of irinotecan on NER gene expression or to an indirect effect on topoisomerase I activity. Complementary studies are required to determine if the cellular parameters identified in this study could be translated at the clinical level to predict clinical response after combined treatment with oxaliplatin and irinotecan in humans.