Association between ERCC1 and XPA expression and polymorphisms and the response to cisplatin in testicular germ cell tumours.

BACKGROUND: Cisplatin cures over 80% of testicular germ cell tumours (TGCTs), and nucleotide-excision repair (NER) modifies the sensitivity to cisplatin. We explored the association between NER proteins and their polymorphisms with cisplatin sensitivity (CPS) and overall survival (OS) of patients with non-seminomatous (ns)-TGCTs. METHODS: The expression of ERCC1 and XPA and the presence of γH2AX were evaluated in cancer cell lines and in fresh ns-TGCTs. The ERCC1 protein was also determined in ns-TGCTs. The differences between CPS and non-CPS cell lines and patients were analysed by Student's t- or χ(2)-tests. The differences in OS were analysed using the log-rank test, and the hazard ratios (HRs) were calculated using the Cox model. RESULTS: High ERCC1 expression was observed in the non-CPS cells, and both ERCC1 and γH2AX expressions were augmented after cisplatin treatment. Increased ERCC1 expression was also identified in non-CPS patients. Neither polymorphism was associated with either CPS or OS. The presence of ERCC1 was associated with non-CPS (P=0.05) and adjusted in the prognosis groups. The HR in ERCC1-negative and non-CPS patients was >14.43, and in ERCC1-positive and non-CPS patients the HR was >11.86 (P<0.001). CONCLUSIONS: High levels of ERCC1 were associated with non-CPS, suggesting that ERCC1 could be used as a potential indicator of the response to cisplatin and prognosis in ns-TGCTs.

Control of skin cancer by the circadian rhythm.

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.

Imiquimod-induced TLR7 signaling enhances repair of DNA damage induced by ultraviolet light in bone marrow-derived cells.

Imiquimod is a TLR7/8 agonist that has anticancer therapeutic efficacy in the treatment of precancerous skin lesions and certain nonmelanoma skin cancers. To test our hypothesis that imiquimod enhances DNA repair as a mechanism for its anticancer activity, the nucleotide excision repair genes were studied in bone marrow-derived cells. Imiquimod enhanced the expression of xeroderma pigmentosum (XP) A and other DNA repair genes (quantitative real-time PCR analysis) and resulted in an increased nuclear localization of the DNA repair enzyme XPA. This was dependent on MyD88, as bone marrow-derived cells from MyD88(-/-) mice did not increase XPA gene expression and did not enhance the survival of MyD88(-/-)-derived bone marrow-derived cells after UV B exposure as was observed in bone marrow-derived cells from MyD88(+/+) mice. Imiquimod also enhanced DNA repair of UV light (UVL)-irradiated gene expression constructs and accelerated the resolution of cyclobutane pyrimidine dimers after UVL exposures in P388 and XS52. Lastly, topical treatment of mouse skin with 5% imiquimod cream prior to UVL irradiation resulted in a decrease in the number of cyclobutane pyridimine dimer-positive APC that were found in local lymph nodes 24 h after UVL irradiation in both wild-type and IL-12 gene-targeted mice. In total, these data support the idea that TLR7 agonists such as imiquimod enhance DNA repair in bone marrow-derived cells. This property is likely to be an important mechanism for its anticancer effects because it protects cutaneous APC from the deleterious effects of UVL.

DNA repair proteins and telomerase reverse transcriptase in the cochlear lateral wall of cisplatin-treated rats.

Cochlear lateral wall damage is a side effect of cisplatin chemotherapy. Recent studies have shown that cisplatin treatment precipitates platinated DNA adducts in the cochlear lateral wall which suggest that DNA damage may contribute to ototoxicity. Platinated adducts are high-affinity substrates for the global genomic nucleotide excision repair (GG-NeR) pathway which is facilitated by xeroderma pigmentosum (Xp) complementing proteins, such as XpC, XpD and XpA. tumor biology has shown that in addition to stimulating GG-NeR, cisplatin may deplete telomerase reverse transcriptase (teRt). in the current study Fischer344 rats were treated with cisplatin (2 mg/kg/4 days, i.p.) and their cochleae harvested for immunohistochemistry. XpC, XpD and XpA expression increased while teRt expression decreased among cisplatin treated animals compared to vehicle control. these findings suggest that in addition to forming platinated adducts, cisplatin chemotherapy may up-regulate DNA repair proteins and modify teRt expression in the cochlear lateral wall.

Dys-synchronous regulation of XPC and XPA in trigeminal ganglion neurons following cisplatin treatment cycles.

BACKGROUND: Cancer cells may survive cisdiamminedichloroplatinum-II (cisplatin) DNA damage through synchronous mobilization of DNA repair proteins such as xeroderma pigmentosum C and A (XPC and XPA). However, non-cancerous neuronal cells exhibit hyper-vulnerability to cisplatin which is manifest as peripheral neurotoxicity. The purpose of the present study was to evaluate the effect of cisplatin on the immunolocalization of XPC and XPA in the trigeminal ganglion. MATERIALS AND METHODS: Fischer344 rats were treated with two cycles of cisplatin (2 mg/kg/day, i.p.) and tissues were harvested for immunohistochemistry after each treatment cycle. RESULT: XPA immunoreactivity was evident after each treatment cycle, however, XPC immunoreactivity was suppressed following the second treatment cycle. CONCLUSION: The hyper-vulnerability of peripheral neurons to cisplatin chemotherapy may relate to the dys-synchronous regulation of XPC and XPA.

Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation.

Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells.

Age at exposure and Apc status influence the levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in mouse intestine and liver.

We have previously shown that C57BL/6J-Min/+ (multiple intestinal neoplasia) mice, heterozygous for the Min mutation in the adenomatous polyposis coli gene, were more susceptible to intestinal tumorigenesis and had higher intestinal PhIP-DNA adduct levels after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) on day 12 than on day 36 after birth [I.-L. Steffensen, H.A.J. Schut, J.E. Paulsen, A. Andreassen, J. Alexander, Intestinal tumorigenesis in multiple intestinal neoplasia mice induced by the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: perinatal susceptibility, regional variation, and correlation with DNA adducts, Cancer Res. 61 (200l) 8689-8696]. In the present study, we have evaluated further whether this difference in susceptibility is related to adduct formation/removal, cell proliferation, apoptosis or expression of the nucleotide excision repair protein Xeroderma pigmentosum group A (XPA) in the intestines. Min/+ and +/+ (wild-type) mice were given a subcutaneous injection of 50 mg/kgbw PhIP on day 12 or 36, and the levels of PhIP-DNA adducts after 8, 12, 24 h, 3 or 7 days were quantified by use of 32P-postlabelling. In Min/+ mice, adduct levels were significantly higher after exposure on day 12 than on day 36 in the middle (1.5- to 8.5-fold) and distal (1.3- to 6.5-fold) small intestine from 8h to 3 days after administration of PhIP, but not in the colon and proximal small intestine. In the liver - a non-target organ for PhIP - adduct levels were 2.0- to 7.5-fold higher after exposure on day 12 than on day 36 from 8 to 24h after exposure. Adduct levels were generally higher in the middle (1.1- to 1.8-fold) and distal (1.1- to 2.0-fold) small intestines of Min/+ compared with +/+ mice after PhIP exposure on day 12, i.e. in the area of the intestines previously found also to have the highest number of tumors in Min/+ mice. PhIP increased cell proliferation and the number of apoptotic cells in the intestine and liver. However, the higher susceptibility to intestinal tumorigenesis in Min/+ mice exposed to PhIP at early age, or in Min/+ mice compared with +/+ mice, could not be explained by differences in cell proliferation, apoptosis or expression of the XPA repair protein.

[Enhancement effect of nucleotide excision repair gene xeroderma pigmentosun group a antisense RNA on sensitivity of human lung adenocarcinoma cell line A549 to cisplatin].

BACKGROUND & OBJECTIVE: Enhanced nucleotide excision repair (NER) capacity is an important mechanism of drug-resistance of tumor cells. Xeroderma pigmentosun group A (XPA) gene plays a key role in early stage of NER pathway. This study was to explore correlation between down-regulation of XPA gene induced by antisense RNA transfection and sensitivity of human lung adenocarcinoma cell line A549 to cisplatin. METHODS: A549 cells were stably transfected with XPA antisense RNA. Positive cell clones were selected by limiting dilution. Northern blot and Western blot were applied to evaluate mRNA and protein levels of XPA in positive cell clones. Sensitivity of A549 cells to cisplatin was evaluated by MTT assay. Host cell reactivation (HCR) assay was used to assess NER capacity of cisplatin-damaged A549 cells. RESULTS: Six positive cell clones, AS1-AS6, were obtained, mRNA and protein levels of XPA were significantly decreased in AS3-AS6 cells. In dose-dependent experiment, the 50% inhibitory concentrations (IC(50)) of cisplatin to parental A549 cells and AS1-AS6 cells were 8.1, 7.6, 4.7, 3.2, 1.9, 2.8, and 4.1 mug/ml, respectively. Sensitivities of AS3-AS6 cells were significantly higher than that of parental A549 cells (F = 9.75, 9.14, 7.39, 8.91u P = 0.005, 0.006, 0.012, 0.006). In addition, mRNA level of XPA was positively correlated with IC(50) of cisplatin (r = 0.927, P = 0.003). Time-effect experiment also showed increases of sensitivity to cisplatin in AS3-AS6 cells. HCR assay showed that NER capacities of AS3-AS6 cells were reduced. When treated with 40, 200, and 1 000 ng/ml of cisplatin, mRNA levels of XPA were positively correlated with cellular NER capacities (r = 0.854, 0.696, 0.858u P = 0.014, 0.082, 0.013). CONCLUSION: The targeted inhibition of XPA by antisense strategy can significantly decrease mRNA level of XPA, reduce cellular NER capacity, and sensitize lung cancer cells to cisplatin.