Dihydrodiol dehydrogenase mediates cisplatin resistance

描述（由申请人提供）： 顺铂是晚期卵巢癌最有效的化疗形式之一，尽管它通常作为卡铂和紫杉醇的组合使用。然而，一些患者由于耐药细胞群的发展而复发。为了鉴定顺铂/卡铂反应性的临床相关标志物，通过微阵列分析和半定量RT-PCR分析了源自亲代人卵巢癌细胞系（2008）的稳定的顺铂耐药人卵巢癌细胞系（2008/C13*）。顺铂耐药细胞中有四个基因表达上调：原肌球蛋白、载脂蛋白J（丛生蛋白）、葡萄糖脱氢酶和二氢脱氢酶。所有四种都被顺铂处理的2008细胞上调。转染实验表明，强迫过表达的只有DDH 1诱导顺铂/卡铂耐药的亲本细胞系。转染后DDH mRNA表达通过RT-PCR监测，蛋白表达通过酶活性测定和免疫组化监测。将DDH 1转染到另外两个人卵巢癌细胞系2780和SKOV 3中导致顺铂卡铂耐药，但对其他抗癌药物不耐药。对具有非常高水平的内源性DDH 2 mRNA的人肺鳞状细胞癌细胞系（A549）的分析显示出大于2008/C13* 细胞系的顺铂抗性程度。我们建议通过（a）转染非卵巢细胞系，包括Tera-2（生殖细胞肿瘤）、A431（宫颈癌细胞）、HTB 56（肺腺癌细胞）和H69（小细胞肺癌细胞）， 显示DDH 1/2表达在源自不同器官的癌症的多种细胞系中产生顺铂卡铂抗性。(b)我们还将尝试确定负责DDH 1上调的转录控制机制以及DDH 1/2产生顺铂抗性的机制（例如，可能涉及活性氧和凋亡途径）。(d)最后，我们将研究人类卵巢癌和肺癌组织化疗前和化疗后（含铂药物）利用单克隆抗体DDH 1/2，以确定DDH表达是否可以作为替代标记物，以确定是否，或不肿瘤将响应铂为基础的化疗。

DESCRIPTION (provided by applicant): Cisplatin is one of the most effective forms of chemotherapy for advanced stage ovarian cancer although it is generally used as a carboplatin taxol combination. However some patients relapse due to the development of a resistant cell population. In order to identify clinically relevant markers for cisplatin/carboplatin responsiveness, a stable cisplatin-resistant human ovarian carcinoma cell line (2008/C13*) derived from the parental human ovarian carcinoma cell line (2008) was analyzed by microarray analysis followed by semiquantitative RT-PCR. Four genes were found to be upregulated in the cisplatin-resistant ceils: tropomyosin, apolipoprotein J (clusterin), glucose dehydrogenase and dihydro dehydrogenase. All four were upregulated by cisplatin treatment of the 2008 cells. Transfection experiments showed that forced overexpression of only DDH1 induced cisplatin/carboplatin resistance in the parental cell lines. DDH mRNA expression posttransfection was monitored by RT-PCR, and protein expression by enzyme activity assays and immunohistochemistry. Transfection of DDH1 into two other human ovarian carcinoma cell lines, 2780 and SKOV3 resulted in cisplatin carboplatin resistance, but not to other anticancer drugs. Analysis of a human lung squamous cell carcinoma cell line (A549) which has a very high levels of endogenous DDH2 mRNA exhibited a degree of cisplatin resistance greater than that of the 2008/C13* cell line. We propose to continue this work by (a) transfection of non-ovarian cell lines, including Tera-2 (germ cell tumors), A431(cervical carcinoma cells), HTB56 (lung adenocarcinoma ceils) and H69 (small cell lung carcinoma cell), to show that DDH1/2 expression produces cisplatin carboplatin resistance in a variety of cell lines derived from cancers from different organs. (b) We will also attempt to determine the transcriptional control mechanisms responsible for DDH1 upregulation as well as the (c) mechanisms by which DDH1/2 produces resistance to cisplatin (e.g. possible involvement of reactive oxygen species and apoptotic pathways involved). (d) Finally, we will study human ovarian and lung carcinoma tissues prior to and post-chemotherapy (with platinum containing drugs) utilizing monoclonal antibodies to DDH1/2 to determine if DDH expression can be used as a surrogate marker to identify whether, or not a tumor will respond to platinum-based chemotherapy.