Investigation of (hypo-)methylation and overexpression of the novel melanoma cell survival gene ASK/Dbf4

S期激酶激活因子（ASK/DBF4）是一个编码细胞分裂周期7 （Cdc7）激活所需的调控亚基的基因（Jackson, Pahl et al. 1993; Yoon, Loo et al. 1993; Sclafani 2000）。ASK/DBF4在正常细胞中表达较差，但在多种癌症和肿瘤细胞系中过表达（Kumagai， Sato等，1999;Nambiar， Mirmohammadsadegh等，2007;Bonte， Lindvall等，2008；Sheu和Stillman 2010）。据报道，ASK/DBF4启动子中的一个611 bp CpG岛包含了主要的转录因子结合位点，代表了一个在正常静止细胞中被推测为高甲基化和抑制的促癌基因，在某些癌症中被推测为低甲基化。我们项目第一阶段的数据表明：(1)正常细胞中ASK/DBF4 mRNA产生的抑制可归因于甲基化介导的直接沉默(2)ASK/DBF4最小启动子甲基化可阻止E2F1介导的ASK/DBF4诱导(3)外胚层谱系癌症中肿瘤进展过程中ASK/DBF4启动子甲基化的患病率显着降低。鉴于人们对降低DNA甲基化的癌症治疗越来越感兴趣，我们的观察结果具有重要意义，因为在某些组织中，基于去甲基化的治疗可能导致发育抑制的原癌基因靶点的再激活（Eden， Gaudet等人2003；Smith， Glazer等人2009）。在项目的第二阶段，我们建议进一步研究(1)在正常细胞和癌细胞中参与谱系特异性ASK/DBF4甲基化/去甲基化的Polycomb抑制复合物组分(2)基因拷贝数变异和DNA去甲基化在ASK/DBF4过表达中的相对贡献。

Activator of S phase kinase (ASK/DBF4) is a gene that encodes a regulatory subunit required for the activation of cell division cycle 7 (Cdc7) (Jackson, Pahl et al. 1993; Yoon, Loo et al. 1993; Sclafani 2000). ASK/DBF4 is expressed poorly in normal cells but is over-expressed in multiple cancers and tumor cell lines (Kumagai, Sato et al. 1999; Nambiar, Mirmohammadsadegh et al. 2007; Bonte, Lindvall et al. 2008; Sheu and Stillman 2010). A 611 bp CpG island in the promoter of ASK/DBF4 encompasses major transcription factor binding sites reported and represented a pro-oncogenic gene that was putatively hypermethylated and repressed in normal quiescent cells and hypomethylated in certain cancers. Our data from the first phase of the project demonstrate that (1) that repression of ASK/DBF4 mRNA production in normal cells can be attributed to direct methylation-mediated silencing (2) ASK/DBF4 minimal promoter methylation prevents E2F1 mediated induction of ASK/DBF4 (3) the prevalence of ASK/DBF4 promoter methylation during tumor-progression decreases significantly in cancers of ectodermal lineage. Our observations gain significant importance in light of the emerging interest in cancer therapies to decrease DNA methylation, as in certain tissues demethylation based therapy may result in reactivation of developmentally repressed proto-oncogene targets (Eden, Gaudet et al. 2003; Smith, Glazer et al. 2009). In the second phase of the project, we propose to further investigate (1) Polycomb repression complex components involved in the lineage specific methylation/demethylation of ASK/DBF4 in normal and cancer cells (2) relative contribution of gene copy number variation and DNA demethylation in ASK/DBF4 over expression.