| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 91 === DNA repair plays a crucial role in the integrity maintenance of the human genome and the prevention of tumorigenesis. Reduced DNA repair capacity has been linked to genetic susceptibility to cancer. Nucleotide excision repair (NER) is a sophisticated DNA repair mechanism that repairs a substantial amount of chemically induced DNA damage and eliminates a wide range of structurally unrelated DNA lesions.
Nonmelanoma skin cancers (NMSC), including basal cell carcinomas (BCC) and squamous cell carcinomas (SCC), have been evidenced with an impaired function in NER. However, malfunction of NER elements in NMSC has not been identified. Xeroderma pigmentosum F (XPF) is an essential subunit in NER and functions as a 5''-incision enzyme when repairing damaged DNA. An adequate expression level of DNA repair genes is essential for normal DNA repair activities. So far, neither XPF''s protein nor antibody is commercially available. To explore the expression of XPF in NMSC, the gene was determined by competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR).
To design feasible primers for the determination of XPF gene''s expression, the complete coding sequences of XPF was folded with DNASIS® software and predicted the mRNA structure. Open regions of the mRNA folding structures were selected for primer synthesis. All the designed primers specifically amplified XPF cDNA as demonstrated by nested PCR, and one set of the primers was mimic constructed to form an internal controlled cDNA for the quantification of XPF gene in NMSC. The exogenous sequences of -Bungarotoxin (-Butx) were designed to share the same XPF primers.
The results indicated that the quantities of XPF expression of BCC and SCC specimens were approximately 57.0% and 76.4% less than that of normal skins, respectively. This paper indicates that the decrease expression of XPF gene may be one of mechanisms for impaired NER in NMSC, and the feasible and quantitative primers used in the experiments may explore the study of XPF in etiology of carcinogenesis.
In the second part of this study, the role of XPF gene on cisplatin resistant of nonsmall cell lung cancer (NSCLC) was investigated. Cisplatin, a DNA damaging agent, is widely used against a broad spectrum of tumors including NSCLC either as a single agent or in combination with other active anticancer drugs. Nevertheless, NSCLC cells were found to be significantly resistant to cisplatin than small cell lung cancer (SCLC). Because mammalian cells remove cisplatin-induced DNA adducts through the nucleotide excision repair (NER) pathway, we proposed that the XPF gene or the entire NER machinery may involve in the resistant of cisplatin in NSCLC.
The cytotoxicity of cisplatin on NSCLC including H441, H520, A549, H661 and H596 cells were evaluated by MTS assay and results indicated that the proliferation of H596 cells was most significantly inhibited by cisplatin; whereas, the others were resistant to cisplatin. Through time-courses study, it is apparent that cisplatin exerts its cytotoxicity effect within 1~4 h in these NSCLC sublines. Platinum (Pt)-DNA adducts were measured by atomic absorption spectrometry (AAS) after cisplatin treatment. The amount of Pt-DNA adducts in H596 cells were significantly higher than H661, H441, H520, A549 cells. The above findings indicate that the cytotoxicity of cisplatin was dependent on the amount of Pt-DNA.
To assess the role of XPF gene in the cisplatin resistant, XPF antisense oligodeoxynucleotides (ODNs) were administrated to verify its effect on cisplatin sensitivity in NSCLC. The synergistic cytotoxicity effect in the combination of XPF antisense ODNs with cisplatin was observed in the H441 and H661 cells; however, H520, A549, and H596 cells were not. Therefore, the causes of cisplatin resistance in H441 and H661 may relate to abnormal XPF gene or NER activity. To further investigate the role of NER in the cisplatin resistant, other NER core factors were examined in this study. Other NER component antisense ODNs target to ERCC1, XPA, RPA and XPG genes were evaluated with cisplatin cotreatment in NSCLC. Results indicated that synergistic effects were observed in cisplatin resistant H441 and H661 cells. It is of interesting that single XPF, ERCC1, XPA, RPA or XPG antisense ODNs administration were resulted in similar results. Apparently, normal NER execution was rely on all core factors participation, defects in any factor were result in abnormal NER function.
This dissertation investigated that the malfunction of XPF gene may result in nonmelanoma skin cancers (NMSC) and its role on the cisplatin resistant in nonsmall cell lung cancer (NSCLC) H441 and H661 cells. By these studies, new strategies may provide in the cancer prevention and therapy.
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