Deficient gene specific repair of cisplatin-induced lesions in Xeroderma pigmentosum and Fanconi's anemia cell lines

Cisplatin is a chemotherapeutic agent known to cause DNA damage.The cytotoxicity of this drug is believed to result from theformation of DNA intrastrand adducts (IA) and DNA interstrandcrosslinks (ICL). While there are many studies on DNA repairof cisplatin damage at the overall level of the genome in varioushuman cell lines, there is little information on the gene-specificrepair. In this report, we have measured the formation and repairof cisplatin induced DNA adducts in the dihydrofolate reductase(DHFR) and ribosomal RNA (rRNA) genes in three cell lines: normalhuman fibroblasts, Fanconi's anemia complementation group A(FAA) and Xeroderma pigmentosum complementation group A (XPA).It is generally thought that XPA cells lack nucleotide excisionrepair and that FAA cells are deficient in the repair of DNAICL. We find that normal human fibroblast cells repair 84% ofthe ICL in the DHFR gene after 24 h, whereas XPA and FAA celllines only repaired 32 and 50% of the ICL respectively. Furthermore,69% of the cisplatin IA in the DHFR gene were repaired in 24h in normal human fibroblasts compared to 22% for XPA and 24%for FAA cells. The repair of the rRNA gene was less efficientthan in the DHFR gene, but the relative pattern between thedifferent cell lines was similar to that of the DHFR gene. Wethus find that FAA cells are deficient not only in the genespecific repair of cisplatin ICL, but also in the gene specificrepair of the more common cisplatin IA. XPA cells are normallythought to be without any nucleotide excision repair capacity,but our data could support a slight ICL unhooking activity.     

Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles

Background  

Testicular germ cell tumors (TGCTs) respond well to cisplatin-based chemotherapy and show a low incidence of acquired resistance compared to most somatic tumors. The reasons for these specific characteristics are not known in detail but seem to be multifactorial. We have studied gene expression profiles of testicular and colon cancer derived cell lines treated with cisplatin. The main goal of this study was to identify novel gene expression profiles with their functional categories and the biochemical pathways that are associated with TGCT cells' response to cisplatin.     

The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines

Background  Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines. Methods  Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC₅₀ and EC₈₀ concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment. Results  Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC₅₀ = 0.27–0.51 μM for cisplatin, 0.52–0.79 μM for oxaliplatin, 0.31–1.26 μM for satraplatin). EC₅₀ values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9–4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed. Conclusions  These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.     

Reversion of 6-thioguanine resistant Chinese hamster cell lines: agent specificity and evidence for the repair of promutagenic lesions

The kinetics and mutagen specificity of reversion of an HGPRT-TG^Rline of Chinese hamster cells have been examined in detail bymeasuring the frequency of HAT^R colonies. Alkylating agentswhich produce relatively high levels of O-atom reaction wereeffective in inducing reversion. MMS, DMS and u.v. were lessefficient, and aflatoxin B₁, acridine orange and N-acetoxy-AAFwere completely ineffective. For agents which were effective,the relationship between HAT^R colony frequency and dose of mutagenwas linear at early expression times (6 h). HAT^R colony frequencyfell subsequently at all doses and the rate and extent of thefall was inversely related to dose. These observations suggestrepair of a pro-mutagenic DNA lesion. Other TG^R mutants isolatedfrom the same wild-type cell line under different selectiveconditions were also tested for revertibility after exposureto the same mutagens. The majority did not revert, this suggeststhat they carry deletions within the structural gene for HGPRT.The infrequent revertible lines all arose spontaneously andour evidence suggests that they carry nonsense mutations.     

DNA interstrand cross-linking and cytotoxicity induced by chloroethylnitrosoureas and cisplatin in human glioma cell lines which vary in cellular concentration of O6-alkylguanine-DNA alkyltransferase.

Fifteen human glioma cell lines were examined for their sensitivity to 1,3-bis(chloroethyl)-nitrosourea (BCNU, carmustine) and cis-dichlorodiamminoplatinum (cisplatin), the induction of DNA interstrand cross-linking (DNA-ISC) induced by the two agents and cellular O6-alkylguanine alkyltransferase (ATase) activity. Cell lines differed in their sensitivities to BCNU by up to 12-fold and to cisplatin by up to 21-fold. For both drugs, the extent of DNA-ISC was related to the drug sensitivity. There was a wide range of cellular ATase levels. Increasing ATase levels correlated with increased resistance to BCNU and with decreased formation of DNA-ISC following treatment with BCNU. In contrast, following treatment with cisplatin, there was no correlation between cellular ATase content and cytotoxicity or between ATase and DNA-ISC. Four sublines of varying ATase activity were prepared from one of the cell lines. These sublines showed a sensitivity to BCNU in inverse proportion to ATase activity, while sensitivity to cisplatin was more uniform. The experiments confirm the direct relationship between ATase concentration and sensitivity to BCNU in glioma cells. Although there was some correlation between cisplatin cytotoxicity and BCNU cytotoxicity, this was not mediated through ATase.     

A comparison of hyperthermia cisplatin sensitization in human ovarian carcinoma and glioma cell lines sensitive and resistant to cisplatin treatment

 Two pairs of human tumor cell lines (glioma and ovarian carcinoma (OvCa)) each having a parental cell line and cisplatin-resistant variant, were evaluated for (a) cisplatin response, (b) hyperthermia response, and (c) combined hyperthermia and cisplatin response. The two resistant lines had comparable resistant responses while for the parental lines, the OvCa was more sensitive than the glioma to cisplatin doses up to 14 μg/ml. For the hyperthermia response, the OvCa parental line was more resistant than the variant line at low-temperature hyperthermia (41° C or 42°C) but became more sensitive at high temperature (45°C). For the glioma, the parental line was more sensitive to hyperthermia at all temperatures tested. Hyperthermia caused sensitization to cisplatin in all cell lines but was generally greater in the glioma cell lines. In the OvCa system, hyperthermia had a slightly greater sensitizing effect on the resistant cell lines, while in the glioma the opposite was true. The degree of sensitization increased with hyperthermia temperature. In summary, the results showed that there is no cross-resistance for hyperthermia and cisplatin, that the degree of thermal sensitization is not reduced in cisplatin-resistant cell lines, and that cisplatin thermal sensitization is cell-line and temperature dependent. Thus, hyperthermia can effectively improve tumor cell response to cisplatin and may be useful in overcoming resistance to cisplatin. Received: 10 March 1995/Accepted: 21 July 1995     

Apoptosis induced by 5-fluorouracil, cisplatin and paclitaxel are associated with p53 gene status in gastric cancer cell lines

In a recent study, we evaluated the efficacy and toxicity of 5-fluorouracil (5-FU), cisplatin (CDDP), and 5-FU plus CDDP (FP treatment) in gastric cancer cell lines and examined the relationship between the response to FP treatment and apoptosis. Our study indicated that there may be prognostic value in measuring p53 prior to FP treatment, and that cancers with wild-type p53 may be better candidates for FP treatment than those with mutant-type p53 in gastric cancer patients. In the present study, because cells with mutant-type p53 are suggested to be less responsive to FP chemotherapy treatment, we examined the relationship between the response to paclitaxel and apoptosis in MKN45 and MKN28 human gastric cancer cell lines by flow cytometry. In both MKN45 and MKN28 cells, paclitaxel arrested cells in the G2/M phases of the cell cycle after 24 h. Additionally, in both MKN45 and MKN28 cells, paclitaxel produced a significant rise in the number of subG1-phase cells after 72 h by the flow cytometry histogram. From these results, our previous study suggests that cells with mutant-type p53 may be less responsive to FP treatment and the present study indicates that another anti-cancer drug like paclitaxel, which might be mediated by a p53-independent pathway, should be selected. These insights may provide a new strategy for gastric cancer chemotherapy, especially second-line chemotherapy or adjuvant chemotherapy.     

Morphological change and cellular differentiation induced by cisplatin in human neuroblastoma cell lines

Summary In 1986 we reported on the capacity ofcis-diamminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell [9]. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 g/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.     

Biological comparison of ovarian cancer resistant cell lines to cisplatin and Taxol by two different administrations

Drug resistance is one of the major obstacles to chemotherapy of ovarian cancer. Studies with cell lines can serve as an initial screen for agents that might modulate drug resistance. To establish more appropriate models of drug resistance and explore whether the differences exist in the different drug resistant sublines selected by different treatments, we induced SKOV3 cell line using cisplatin (CDDP) and Taxol over a period of 16 months by the pulse (SKOV3/CDDP-P and SKOV3/Taxol-P) and intermittent incremental (SKOV3/CDDP-80 and SKOV3/Taxol-25) method, respectively. The resistant phenotype of the four resistant sublines, SKOV3/CDDP-P, SKOV3/CDDP-80, SKOV3/Taxol-P and SKOV3/Taxol-25, was very stable and the resistance index was 4.12, 11.50, 261.98 and 622.76, respectively. In cell morphology, the cells from pulse treatment had remarkable changes compared with the cells from intermittent incremental treatment. SKOV3/CDDP-80 and SKOV3/Taxol-P grew more slowly than SKOV3/CDDP-P and SKOV3/Taxol-25. Multidrug resistance gene 1, multidrug resistance protein 1, lung resistance protein and glutathione S-transferase pi mRNA expression of SKOV3/CDDP-P and SKOV3/Taxol-25 had greater changes than that of SKOV3/CDDP-80 and SKOV3/Taxol-P. The results suggest there are great differences between the resistant cell lines resulting from pulse and intermittent incremental method. The resistant cells selected by the intermittent method were more resistant than the cells selected by the pulse method. The two resistant sublines selected by the pulse method may serve as appropriate models for the study of mechanisms of drug resistance in ovarian cancer.     

Sequence specificity of mutation induced by the anti-tumor drug cisplatin in the CHO aprt gene

Cis-diammine dichloroplatinum (cisplatin) is an effective anticancerdrug which forms adducts with DNA, in both bacterial and mammaliancells. It is suspected of producing tumors as well. To determinethe molecular nature of geneti alterations induced by cisplatin,we cloned and sequenced cisplatin-induced mutants in the adeninephosphoribosyl-transferase (aprt) gene of Cinese hamster ovary(CHO) cells. Mutation by cisplatin appears to be targeted asthe sites of mutation are consistent with the known bindingspecificity of cisplatin. Many mutations occur at or proximalto the sequence 5'-AGG-3' and 5'-GAG-3' and include transversions,transitions, frameshifts and short deletions and duplications.Several double changes were also observed. No major rearrangementswere recovered in our collection. At several locations, a numberof mutants were found to be clustered within a small targetregion, but unlike traditional hotspots, tese represent diversechanges occurring in a localized region of a few base pairs.     

Gene expression in X-irradiated human tumour cell lines expressing cisplatin resistance and altered DNA repair capacity

Previous studies have indicated that excision repair genes,such as ERCC1, or early response genes, such as c-fos, may playa significant role in regulating cellular responses to cisplatin(CDDP) by mediating DNA synthesis and repair pathways. Thispresent study aimed to determine whether altered gene expressionmediated CDDP resistance expressed in two human tumour sublinesfollowing their in vitro exposure to fractionated X-irradiation,not to the drug itself. These sublines, designated SuSa/DXR10and SKOV-3/DXR10, established respectively from a testicularteratoma cell line (SuSa) or an ovarian carcinoma cell line(SKOV-3), expressed stable 3.1- and 2-fold levels of CDDP resistance,as judged by clonogenic assay. Both sublines expressed c-fos,c-myc and thymidylate synthase (TS) RNA constitutively, butat comparable levels to their parental counterparts. Whilstthe ovarian carcinoma cells inherently expressed markedly higherlevels (30- to 50-fold) of the excision repair gene ERCC1 thanthe teratoma cells, only the teratoma DXR10 subline showed anincreased level of expression of ERCC1 mRNA relative to theirparental cells. Expression of the ERCC3/XPB gene encoding arepair helicase, however, was similar in all the lines tested.The results suggest that CDDP resistance may be mediated bydifferent mechanisms in these DXR10 sublines from those previouslyidentified in drug-selected CDDP-resistant human ovarian A2780/DDPcells.     

Activity of a trinuclear platinum complex in human ovarian cancer cell lines sensitive and resistant to cisplatin: cytotoxicity and induction and gene-specific repair of DNA lesions

A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N- ras) level were observed between BBR 3464 and CDDP.     

耐药相关基因二氢叶酸还原酶在人胰腺癌细胞株中的表达

目的探讨耐药相关基因二氢叶酸还原酶（DHFR）在胰腺癌细胞株中的表达。方法通过RT—PCR和Western-blot方法分别测定六株人胰腺癌细胞（SW1990,Capan-1,AsPC-1,MiAPaCa-2,PANC-1,P3）中的DHFR在基因和蛋白水平上表达。结果在六株人胰腺癌细胞株中,DHFR在基因水平上的表达各细胞株之间没有显著性差异（P〈0．05）;DI-IFR蛋白的表达水平以AsPC-1和P3较高,与其余四株细胞之间均存在着显著性差异（P〈0．05）,而SW1990、Capan-1、MiAPaCa-2、PANC-1之间及AsPC-1、P3之间则无显著性差异（P〉0．05）。结论结合本组先前研究结果提示胰腺癌细胞株对DHFR抑制剂的低敏感性与DHFR蛋白的高水平表达有关,DHFR参与了胰腺癌对化疗的耐药。     

Effect of novobiocin on cisplatin cytotoxicity and dna interstrand cross-link formation in a cisplatin-resistant,small-cell lung carcinoma cell line

Studies were performed to determine whether novobiocin can be used to enhance cisplatin (CDDP) cytotoxicity in a human small-cell lung carcinoma cell line, GLC₄/CDDP, resistant to CDDP. Continuous incubation with novobiocin enhanced the cytotoxicity of CDDP treatment 1.9-fold in the parental cell line GLC₄, but had no effect on its cytotoxicity in the resistant cell line GLC₄/CDDP. Short incubation with novobiocin enhanced the cytotoxicity of CDDP treatment in GLC₄ and GLC₄/CDDP by a factor of 4.1 and 2.8, respectively. Using the latter schedule, the amount of CDDP-induced DNA interstrand cross-links (DNA ISC) at 4 hr as well as at 24 hr after novobiocin and CDDP treatment was higher in GLC₄ than in GLC₄/CDDP. In this case, the amount of DNA ISC had increased 1.6-fold in GLC₄and 1.3-fold in GLC₄/CDDP at 4hr, and 2.7-fold and 1.4-fold, respectively, in these cell lines at 24 hr after treatment compared to CDDP treatment alone. Our results suggest an effect of novobiocin on the formation of DNA ISC. The decreased efficacy of novobiocin, an inhibitor of DNA topoisomerase (Topo) II catalytic activity, in GLC₄/CDDP may be due to the increased Topo II activity previously found in the resistant cells. In the present study, we showed that increased Topo II activity was not due to changes in amounts of Topo II in nuclei or nuclear extracts of GLC₄/CDDP. Further analysis of the chromatin, that includes Topo II, showed that the chromatin in nuclei of GLC₄/CDDP was more sensitive to micrococcal nuclease digestion than GLC₄. In addition, the amount of a 56-kDa protein was increased 2-fold in nuclei and nuclear matrices from GLC₄/CDDP. The reduced efficacy of novobiocin to increase the CDDP cytotoxicity as well as the formation of DNA ISC in GLC₄/CDDP compared to GLC₄ may be due to changes in the chromatin structure of the resistant cells.     

Combination effect of caffeine and cisplatin on a cisplatin resistant human lung cancer cell line

The development of resistance to anti-cancer drugs is a major factor in limiting the response rate of cancer chemotherapy. Several mechanisms of such resistance are reported. Recently, expression of MDR gene and synthesis of p-glycoprotein by the MDR gene was reported as a mode of multi-drug resistance, but the mechanism of the resistance to cisplatinum (CDDP) remains unclear. Detoxification of CDDP, increase of the efflux of the drug and increase of DNA repair are considered to be the mode of CDDP resistance. It is widely documented that caffeine enhances the cytocidal effect of certain anti-cancer agents. The inhibition of DNA repair by caffeine has been considered to be one mechanism which enhances the cytocidal effect of such agents. We conducted the present study to evaluate the combination effect of caffeine and CDDP on the human lung adenocarcinoma cell line PC9/P and its CDDP resistant cell line PC9/R. Cell growth inhibition was measured by clonogenic assay and cell cycle analysis was performed with propidium iodide (PI) stain using flow cytometer (FCM). Caffeine enhanced the effect of CDDP on PC9/P synergistically. However, the combination effect of the two drugs was not apparent on PC9/R. Caffeine decreased G2M accumulation due to CDDP exposure in both cell lines. The data indicate that caffeine does not overcome the resistance of PC9/R, whereas caffeine enters PC9/R. It is suggested that increase of DNA repair might not be a mode of the CDDP resistance of PC9/R.     

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.     

Repair analysis of 4-hydroperoxycyclophosphamide-induced DNA interstrand crosslinking in the c-myc gene in 4-hydroperoxycyclophosphamide-sensitive and-resistant medulloblastoma cell lines

Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, development of resistance to this alkylator frequently occurs and is the harbinger of tumor progression and death. In order to understand the biochemical basis of this resistance, we generated a panel of medulloblastoma cell lines in our laboratory that were resistant to 4-hydroperoxycyclophosphamide (4-HC). Previously, we have shown that elevated levels of aldehyde dehydrogenase and glutathione mediate cellular resistance to 4-HC. The present study was conducted to identify the third unknown mechanism mediating the resistance of cell line D283 Med (4-HCR) to 4-HC, testing the hypothesis that this resistance is mediated by an increased repair of DNA interstrand crosslinks (ICLs). The doses of 4-HC that produced a one- and two-log cell kill of D283 Med cells were 25 and 50 M, respectively, compared with values of 125 and 165 M in D283 Med (4-HCR), the resistant cell line. The formation and disappearance of 4-HC-induced DNA ICLs at the c-myc gene were subsequently studied by DNA denaturing/renaturing gel electrophoresis and Southern blot analysis. 4-HC-induced DNA ICLs in the c-myc gene exhibited a dose-dependent relationship. The percentage of the c-myc gene that was crosslinked was approximately 1–3% at a dose of 100 M. More than 50% of the DNA crosslinking in D283 Med (4-HCR) cells was removed by 6 h after drug treatment, whereas, in D283 Med cells, more than 90% of the DNA crosslinking was still present at 6 h. These findings suggest that the increased repair of DNA ICLs in D283 Med (4-HCR) may contribute significantly to its resistance to 4-HC.This work was supported by NIH Grants CA 11898, CA 56115, NS 30245, NS 20093; and ACS Grant DHP-67E     

Differential effects of cholesterol and phytosterols on cell proliferation, apoptosis and expression of a prostate specific gene in prostate cancer cell lines

Background: The purpose of our study was to show the apoptotic and anti-proliferative effects of phytosterols as distinct from cholesterol effects on prostate cancer cell lines, and also their differential expression of caveolin-1, and a prostate specific gene, PCGEM1. Methods: PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 48 h, followed by trypan blue dye exclusion measurement of cytotoxicity and MTT cell proliferation assays, respectively. Cell cycle analysis was carried out microscopically, and by propidium iodide uptake using flow cytometry. Sterol induction of oncogenic gene expression was evaluated by RT-PCR. Apoptotic cells were identified by immunocytochemistry using DNA fragmentation method, and by annexin V adhesion using flow cytometry. Results: Physiological doses (16 μM) of these sterols were not cytotoxic in these cells. Cholesterol-enrichment promoted mitosis (54 and 61% by microscopy; 40.8 and 34.08% by FACS analysis in PC-3 and DU145, respectively) and cell growth (P < 0.05), while phytosterols suppressed mitosis (29 and 35% by microscopy; 27.71 and 17.37% by FACS analysis in PC-3 and DU145, respectively), and significantly induced tumor-suppression (P < 0.05) and apoptosis. We demonstrated for the first time that cholesterols upregulated the expression of PCGEM1 even in androgen-insensitive prostate cancer cell lines. Phytosterols reversed this effect, while upregulating the expression of caveolin-1, a known mediator of androgen-dependent proto-oncogene signals that presumably control growth and anti-apoptosis. Conclusions: Phytosterol inhibition of PCGEM1 and cell growth and the overexpression of caveolin-1, suggests that poor disease prognosis anchors on the ability of caveolin-1 to regulate downstream oncogene(s) and apoptosis genes. Sterol intake may contribute to the disparity in incidence of prostate cancer, and elucidation of the mechanism for modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or chemotherapeutic intervention. Sterol regulation of PCGEM1 expression suggests its potential as biomarker for prediction of neoplasms that would be responsive to chemoprevention by phytosterols.