Fluorescent annulated imidazo[4,5-c]isoquinolines via a GBB-3CR/imidoylation sequence - DNA-interactions in pUC-19 gel electrophoresis mobility shift assay.

Herein we report the development of a sequential synthesis route towards annulated imidazo[4,5-c]isoquinolines comprising a GBB-3CR, followed by an intramolecular imidoylative cyclisation. X-Ray crystallography revealed a flat 3D structure of the obtained polyheterocycles. Thus, we evaluated their interactions with double-stranded DNA by establishing a pUC-19 plasmid-based gel electrophoresis mobility shift assay, revealing a stabilising effect on ds-DNA against strand-break inducing conditions.

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours.

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.

DNA repair in cisplatin-sensitive and resistant human cell lines measured in specific genes by quantitative polymerase chain reaction.

More than 80% of patients with testicular germ cell tumours (TGCT) are cured using cisplatin-based combination chemotherapy, and resistance to cisplatin is the final barrier to the cure of nearly all patients with this disease. In this study, we used quantitative polymerase chain reaction (Q-PCR) to investigate the role of DNA repair in cisplatin resistance in two genes, one transcribed and one not transcribed. Three pairs of cisplatin-sensitive and resistant cell lines were used, two derived from TGCT and one from a bladder cancer. In these pairs of sublines, we observed no major differences between the repair of cisplatin-induced damage in the transcribed and nontranscribed genes, nor did there appear to be any relationship between DNA repair capacity and the development of cisplatin resistance. Despite the strong indication that the sensitivity of testis tumour cells to cisplatin is related to their reduced ability to repair cisplatin-damaged DNA, these cells apparently do not become resistant to cisplatin by acquiring DNA repair capacity.

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs.

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.

Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair.

The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations.

Molecular characterization of methyl methanesulphonate (MMS)-induced HPRT mutations in V79 cells.

Spontaneous and methyl methanesulphonate-induced HPRT-deficient mutants were analysed for changes in the hprt gene structure using multiplex polymerase chain reaction. The PCR amplification pattern of 21 MMS-induced mutations revealed one total deletion of the hprt coding exons and one small deletion within exon 5, while 19 mutants showed the V79 wild-type pattern. Molecular analysis of 30 spontaneous mutations revealed no mutants with amplification patterns which differed from those of wild-type cells. We further analysed MMS-induced mutants in a different V79 cell line with a high (40%) spontaneous deletion frequency. MMS caused a dose-dependent increase in the mutant frequency but the incidence of deletions was reduced to 6% at 2 x 10(-4) M and to 13% at 5 x 10(-4) M indicating that mainly point mutations were induced. The repair inhibitor cytosine arabinoside (araC) enhanced mutation induction by MMS but did not change the proportion of deletions in the mutation spectrum. The results indicate that different V79 cell lines spontaneously produce different amounts of deletion mutations. The frequency of MMS-induced deletions does not depend on the frequency of spontaneous deletions in a given cell line. The MMS-induced mutation spectrum seems to be unchanged even at high concentrations with a strong cytotoxic effect. Deletions are not increased as a consequence of araC-inhibited repair of MMS-induced lesions.

Cytogenetic and molecular characterization of the mutagenicity of chlorambucil in V79 cells.

Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. Recently, it could be shown that CBC is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes. We therefore reinvestigated the in vitro genotoxic effects of CBC in V79 cells and characterized induced sister-chromatid exchanges (SCEs), chromosome aberrations and gene mutations by means of cytogenetic and molecular methods. CBC effectively induced chromosome aberrations and SCEs in a dose-dependent manner. The chromosome aberrations found after a 14-h treatment were mainly chromatid-type aberrations. 3-Aminobenzamide (3AB) did not influence the incidence of CBC-induced SCEs and chromosome aberrations. Combined treatment with CBC and caffeine (CAF) strongly increased the frequency of aberrations, but had no effect on the yield of SCEs. CAF at lower concentrations enhanced the production of chromatid breaks and exchange figures while higher concentrations (10(-3) M) caused multiple breaks and pulverised mitoses. Mutations at the hprt locus were induced in a narrow range of CBC concentrations (10(-5) M-2 x 10(-5) M) and the mutagenic effect was accompanied by strong cytotoxicity. The CBC-induced gene mutation frequency was not increased after CAF treatment. The molecular analysis of CBC-induced mutations by Southern hybridization and PCR demonstrated that CBC predominantly produced small alterations but not deletions or gross structural alterations in the hprt gene of V79 cells. For the first time, these results reveal striking differences in the mutagenic action of an alkylating agent in cultivated cells compared to germ-line cells at the molecular level.

Exceptional sensitivity of testicular germ cell tumour cell lines to the new anti-cancer agent, temozolomide.

Metastatic testicular germ cell tumours are cured in approximately 85% of patients using cisplatin-based combination chemotherapy. Patients who fail to respond have a poor prognosis, and there is a need for more effective treatments for cisplatin-resistant disease. In this study, it is shown that two of four cell lines derived from human non-seminomatous testicular germ cell tumours are exceptionally sensitive to temozolomide, a new imidazotetrazine which can cross the blood-brain barrier in mice. In addition, three pairs of cisplatin-resistant sublines show little cross-resistance to temozolomide. These data suggest that temozolomide might have activity against non-seminomatous testicular germ cell tumours which have relapsed following cisplatin-containing chemotherapy, and could have a role in the treatment of patients with metastatic lesions in the brain.

Genetic effects of 2-aminopurine in mammalian cells.

The effects of 2-aminopurine (APur) on mutations, sister-chromatid exchanges (SCEs) and proliferation were investigated in V79 cells by means of cytogenetic and flowcytometric experiments. APur did not induce SCEs after a 3-h treatment before the addition of BrdUrd but SCE frequencies were increased after treatment for two cell cycles in the presence of BrdUrd. SCEs were mainly produced during the second cell cycle of the SCE experiment when BrdUrd substituted DNA is replicated. APur also caused a high percentage of polyploid cells. Compared on the basis of DNA content, SCE induction was the same in diploid and tetraploid metaphases. APur-induced SCEs are strongly influenced by nucleosides. The presence of deoxycytidine (dCyd) caused a reduction of AP-induced SCEs to about control level while addition of deoxythymidine (dThd) enhanced SCE induction. Flow cytometric measurements revealed a small increase in S-Phase cells and a strong accumulation in G2/M after APur treatment in the presence of BrdUrd. S-phase delay was strongly enhanced when BrdUrd substituted DNA is replicated. Addition of dCyd removed the APur-induced inhibition of S-phase in both protocols. Using the same treatment protocol, APur also induced mutations at the HPRT locus. In contrast to their effects on SCEs and proliferation neither BrdUrd nor dCyd had an effect on APur-induced mutations, and dThd reduced the mutation frequency. The results demonstrate that APur-induced SCEs and mutations occur independently from each other. APur-induced mutations obviously occur by a mispairing mechanism while SCEs are a consequence of pool imbalances during replication.

Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction.

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.

Gene-specific repair of Pt/DNA lesions and induction of apoptosis by the oral platinum drug JM216 in three human ovarian carcinoma cell lines sensitive and resistant to cisplatin.

JM216, an oral platinum drug entering into phase III clinical trial, exhibited comparable cytotoxicity to cisplatin in three human ovarian carcinoma cell lines: the sensitive (CH1), acquired resistant (CH1cisR) and intrinsically resistant (SKOV-3). Platinum accumulation and binding to DNA were similar in each of the three cell lines at equimolar doses, indicating that the resistant cell lines could tolerate higher intracellular platinum levels and platinum bound to DNA at IC50 concentrations of drug. Comparison with cisplatin demonstrated that intracellular platinum levels were marginally higher with JM216, but that platinum binding to DNA was similar for the two drugs in each of the cell lines. Each of the cell lines exhibited an ability to repair JM216 induced platinum/DNA lesions in the N-ras gene (gene-specific repair) at equitoxic concentrations of drug. However, this occurred to a greater extent in the two resistant cell lines such that by 24 h the CH1cisR and SKOV-3 had removed 72% and 67% respectively compared with approximately 32% for the CH1. Reduced gene-specific repair capacity in CH1 cells was also seen following incubation with 25 microM (or 5 microM - 2 x IC50) cisplatin, whereas the CH1cisR and SKOV-3 cell lines were repair proficient. JM216 induced apoptosis in the three cell lines following a 2h incubation with 2 x the IC50 of drug. Fluorescent microscopy of cells stained with propidium iodide showed that the detached cell population displayed typical apoptotic nuclei. Furthermore, field inversion gel electrophoresis demonstrated the presence of DNA fragments approximately 23-50 kb in size, indicative of apoptosis, in the detached cells. JM216 induced an S phase slow down in each of the three cell lines accompanied by a G2 block in the CH1 pair. Incubation with this concentration of JM216 also resulted in the induction of p53 in the CH1 and CH1cisR. These studies suggest that the relative sensitivity of the CH1 cell line to cisplatin and JM216 is at least partly attributable to a deficiency in gene-specific repair. The oral platinum drug, JM216, exerts its cytotoxic effects through the induction of apoptosis following a slow-down in S phase in both the sensitive and resistant lines.

Reactivity of germ cell maturation stage-specific markers in spermatocytic seminoma: diagnostic and etiological implications.

It is generally accepted that testicular seminomas and spermatocytic seminomas have separate pathogeneses, although the origin of these two types of germ cell tumors of the adult testis remains a matter of debate. Although an embryonic germ cell origin seems to be most likely for seminomas, a spermatogonia-spermatocyte origin has been suggested for spermatocytic seminoma. To shed more light on the etiology of spermatocytic seminomas, we undertook an immunohistochemical and molecular approach using SCP1 (synaptonemal complex protein 1), SSX (synovial sarcoma on X chromosome), and XPA (xeroderma pigmentosum type A) as targets. Although a stage-specific expression pattern has been reported for SCP1 and SSX in normal spermatogenesis, we demonstrate here that it also exists for XPA. In fact, immunohistochemistry shows that the proteins of SCP1 and XPA are specifically present in the stage of primary and pachytene spermatocytes. In contrast, SSX was found in spermatogonia and primary spermatocytes, as well as in germ cells, from at least the 17th week of intrauterine development onward. Although no protein encoded by any of these genes was detected in tumor cells of a series of testicular seminomas, all tested spermatocytic seminomas were positive, in agreement with expression analysis. These data support the model that seminomas originate from an embryonic germ cell, and they imply that the cell of origin of spermatocytic seminomas is at least capable of maturing to the stage of spermatogonia-pachytene spermatocyte.

DNA repair capacity and cisplatin sensitivity of human testis tumour cells.

Metastatic testicular germ cell tumours can be cured using cisplatin-based combination chemotherapy. To investigate the role of DNA repair in cisplatin sensitivity, we measured the formation and removal of cisplatin adducts in the whole genome and in specific genomic regions in 3 testis and 3 bladder cancer cell lines. Following a 1 hr exposure to 50 microM cisplatin, the mean level of DNA platination was lower in the testis tumour cell lines. During a 72 hr post-treatment incubation period, the 3 bladder cell lines removed platinum from the overall genome, whereas 2 of the testis tumour cell lines showed relatively little reduction of DNA platination. The third testis tumour cell line, SuSa, showed an intermediate capacity to remove cisplatin. Cisplatin-induced damage and repair in selected regions of the actively transcribed N-ras gene and the inactive CD3delta gene were measured using quantitative PCR. The data were in agreement with those obtained with atomic absorption spectroscopy for the whole genome, showing that the bladder lines were repair-proficient: 2 of the testis tumour cell lines showed no repair, and the third testis line, SuSa, showed an intermediate level of repair in these 2 genes. Our findings confirm that reduced capacity to repair cisplatin-damaged DNA may contribute to the hypersensitivity of testis tumour cells to DNA-damaging agents.