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，尽管它通常被用作卡铂紫杉醇的联合用药。然而，由于耐药细胞群的发展，一些患者会复发。为了寻找顺铂/卡铂敏感性的临床相关标记物，以人卵巢癌细胞系(2008)为研究对象，采用基因芯片技术和半定量逆转录聚合酶链式反应(RT-PCR)技术对人卵巢癌细胞系(2008/C13*)进行了分析。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(Clusterin)、葡萄糖脱氢酶和二氢脱氢酶。顺铂处理2008年的细胞后，这四种基因的表达均上调。转染实验表明，强制过表达DDH1仅诱导亲本细胞对顺铂/卡铂耐药。逆转录聚合酶链式反应(RT-PCR)检测DDH基因的表达，酶活性分析和免疫组织化学方法检测蛋白表达。将DDH1基因导入另外两株人卵巢癌细胞株2780和SKOV3中，可产生对顺铂、卡铂的耐药性，但对其他抗癌药物无耐药性。对内源性DDH2 mRNA水平非常高的人肺鳞状细胞癌细胞系(A549)的分析显示，与2008/C13*细胞系相比，顺铂的耐药性程度更高。我们建议通过以下方法继续这项工作：(A)将包括Tera-2(生殖细胞肿瘤)、A431(宫颈癌细胞)、HTB56(肺腺癌细胞)和H69(小细胞肺癌细胞)的非卵巢细胞系 结果表明，DDH1/2的表达在来自不同器官的癌症来源的各种细胞系中产生顺铂卡铂耐药性。(B)我们还将尝试确定导致DDH1上调的转录调控机制，以及(C)DDH1/2产生顺铂耐药的机制(例如，可能参与的活性氧和涉及的细胞凋亡途径)。(D)最后，我们将利用DDH1/2的单抗研究化疗前和化疗后(含铂药物)的人卵巢癌和肺癌组织，以确定DDH的表达是否可以作为替代标记物来确定肿瘤是否对以铂为基础的化疗有反应。

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.