Phenotypic reversion of cisplatin resistance in human cells accompanies reduced host cell reactivation of damaged plasmid

Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance.     

Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways

The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.     

Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.     

Two-wavelength fluorescence assay for DNA repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.     

Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) have proven to be essential elements in many aspects of DNA metabolism including replication, repair and recombination. We have developed an in vitro assay in which the presence of an interstrand crosslink stimulates the incorporation of radiolabeled nucleotides into both damaged and undamaged plasmid DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent inhibitor of PCNA, was found to strongly inhibit crosslink-induced incorporation. Addition of exogenous PCNA partially restored the resynthesis activity. Likewise, neutralization of RPA by monoclonal antibodies also inhibited incorporation, but the effect was somewhat more pronounced on the undamaged plasmid than the damaged plasmid. Addition of excess RPA also partially reversed antibody inhibition. These results indicate that both PCNA and RPA are required for efficient in vitro DNA resynthesis induced by interstrand crosslinks.     

The influence of folic acid depletion on the Nucleotide Excision Repair capacity of human dermal fibroblasts measured by a modified Host Cell Reactivation Assay

Animal and human studies have shown that low levels of folic acid are associated with an impaired DNA Repair Capacity (DRC) and an increased cancer risk. However, the molecular evidence that folic acid enhances the DRC of cultured human cells is still limited because of a paucity of in vitro studies. We investigated the effect of folic acid depletion in vitro on the DRC of human dermal fibroblasts derived from 17 donors of different ages. To assess the cellular Nucleotide Excision DRC, we used a modified Host Cell-Reactivation Assay (HCRA), adapted to the Fluorescence Activated Cell Sorting (FACS)-technology, which is highly sensitive in comparison to luminometer-technology and allows single cell based analysis. We used DsRed as a reporter (irradiated with UVC light) and pEGFP to control the performance of the transformations. Folic acid had a statistically significant effect on the DRC in all of the 17 donors, however, the levels varied considerably between individuals (2.0-19.6%). When the effect of folic acid substituted on the DRC was compared to donor age, we observed that there was less DNA repair in old donors compared to the younger donors, although this was only significant at lower levels.     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.Key words: DNA methylation, lymphoblastoid cell lines, peripheral blood leukocytes, LUMA, promoter tiling array, gene expression     

The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: a pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O⁶-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.     

Wortmannin增强细胞辐射敏感性的分子机制

 Ｗ ｏｒｔｍ ａｎｎｉｎ（ Ｗ Ｏ Ｒ Ｔ）是 Ｐ Ｉ３ Ｋ 家族激酶特异抑制剂,对 ｐ５３ 野生型及突变型细胞的辐射敏感性均有提高．为了阐明 Ｗ Ｏ Ｒ Ｔ 的辐射增敏机制,通过免疫沉淀及免疫印迹法分析了 Ｗ Ｏ Ｒ Ｔ对辐射引起的细胞 Ｇ２／ Ｍ 转换中关键分子 ｃｄｃ２ 酪氨酸脱磷酸化延迟的影响;通过流式细胞术分析了 Ｗ Ｏ Ｒ Ｔ 对辐射引起的细胞 Ｇ２ 期延迟、细胞凋亡的影响;经报告基因转染的方法分析了 Ｗ Ｏ Ｒ Ｔ对宿主细胞对辐射损伤报告基因修复的影响;发现 Ｗ Ｏ Ｒ Ｔ 可促进受照细胞 ｃｄｃ２ 酪氨酸脱磷酸化、减弱辐射引起的细胞 Ｇ２ 期延迟、增强细胞凋亡并抑制损伤 Ｄ Ｎ Ａ 修复．提示 Ｗ Ｏ Ｒ Ｔ 辐射增敏是通过干扰细胞 Ｇ２ 期检查点调控、抑制损伤 Ｄ Ｎ Ａ 修复和促进细胞凋亡等多种途径实现的．     

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60J/m(2). NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.     

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.     

Effect of dithiocarbamate pesticide zineb and its commercial formulation,azzurro. IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 microg/ml) or azzurro (100.0 microg/ml) for 80min, washed and reincubated in pesticide-free medium for 0-12h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6h of incubation. After 12h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0-12h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5-12h repair time for both pesticides. At 12h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0-12h repair period was observed.     

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.     

X射线对人外周血淋巴细胞DNA损伤修复和凋亡的影响

目的 研究不同时间诱导X射线照射的淋巴细胞进入细胞周期DNA损伤修复与凋亡的影响.方法 X射线（0.5 Gy）作用于正常人外周血淋巴细胞,以照射后不同时间点（0、4 h）分别加入PHA并分成两组,即照射后0 h加PHA组（A组）和照射后4 h加PHA组（B组）,再分别培养0、0.5、2 h,用流式细胞术和免疫印迹法检测A组和B组γ-H2AX蛋白的表达,Annexin-V/PI法分析A、B两组的细胞凋亡率.结果 流式细胞术及免疫印迹结果均显示A组的γ-H2AX蛋白表达高于B组（P＜0.05）,且均先升高后降低.A组细胞凋亡率亦大于B组.结论 不同时间诱导被打击的淋巴细胞进入周期其可能发生DNA修复并同时伴随细胞凋亡的发生.     

Comparative analysis of DNA methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

Previous reports have shown that DNA methylation profiles within primary human malignant tissues are altered when these cells are transformed into cancer cell lines. However, it is unclear if similar differences in DNA methylation profiles exist between DNA derived from peripheral blood leukocytes (PBLs) and corresponding Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs). To assess the utility of LCLs as a resource for methylation studies we have compared DNA methylation profiles in promoter and 5/ regions of 318 genes in PBL and LCL sample pairs from patients with type 1 diabetes with or without nephropathy. We identified a total of 27 (~8%) genes that revealed different DNA methylation profiles in PBL compared with LCL-derived DNA samples. In conclusion, although the profiles for most promoter regions were similar between PBL-LCL pairs, our results indicate that LCL-derived DNA may not be suitable for DNA methylation studies at least in diabetic nephropathy.     

DNA repair excision nuclease attacks undamaged DNA. A potential source of spontaneous mutations

Nucleotide excision repair is a general repair system that eliminates many dissimilar lesions from DNA. In an effort to understand substrate determinants of this repair system, we tested DNAs with minor backbone modifications using the ultrasensitive excision assay. We found that a phosphorothioate and a methylphosphonate were excised with low efficiency. Surprisingly, we also found that fragments of 23-28 nucleotides and of 12-13 nucleotides characteristic of human and Escherichia coli excision repair, respectively, were removed from undamaged DNA at a significant rate. Considering the relative abundance of undamaged DNA in comparison to damaged DNA in the course of the life of an organism, we conclude that, in general, excision from and resynthesis of undamaged DNA may exceed the excision and resynthesis caused by DNA damage. As resynthesis is invariably associated with mutations, we propose that gratuitous repair may be an important source of spontaneous mutations.     

Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.