HMG-1/Y AND NEOPLASTIC TRANSFORMATION

描述：（改编自研究者摘要）正常细胞 增殖是由生长因子启动的，这些生长因子结合到它们的特异性 受体，并最终导致基因的顺序表达 在调节细胞生长中起重要作用。 虽然许多细胞周期 调控基因已被确定，进一步阐明这种遗传 该计划将提高我们对正常和肿瘤细胞生长的理解。 即刻早期基因在生长因子 刺激，包括c-myc原癌基因;延迟早期基因， 在生长因子刺激后数小时激活，但在DNA合成之前， 并包括HMG-I/Y基因。 为了更好地理解 延迟早期基因在细胞周期的G1/S转换中被激活， 我们一直在研究HMG-I/Y基因的调控。 他们的实验室 HMG-I/Y是c-Myc的基因靶点。 他们的 初步结果还表明，HMG-I同种型单独引起 实验细胞系中的肿瘤转化。 由于表达式 c-myc和HMG-I/Y的表达与细胞增殖相关， 肿瘤转化，我们假设HMG-I/Y是重要的c-Myc c-Myc所需的靶基因，用于调节正常和肿瘤 细胞生长 此外，HMG-I/Y在癌细胞中表达增加， 不以c-myc表达升高为特征的细胞系。 因此， 提出HMG-I也可能导致肿瘤转化，独立于 c-Myc的 因此，本研究提案的主要重点将是定义 HMG-I/Y在肿瘤转化中的作用。 本文概述了具体的目标和基本的实验方法， 建议包括：1）评价HMG-I/Y在转化细胞系中的作用 其特征在于增加的c-myc和/或HMG-I/Y表达。 反义和 将潜在的显性阴性HMG-I/Y载体转染入 合适的细胞系，并进行软琼脂转化试验， 解决这个目标。 2)确定HMG-I/Y是否在诱导的细胞凋亡中起作用 c-myc 反义和潜在的显性阴性HMG-I/Y载体将 也可以使用。 此外，我们将确定是否异位表达的 HMG-I/Y导致细胞凋亡。 3)研究HMG-I的机制 导致肿瘤转化。 通过突变分析，我们将 鉴定细胞转化所需的HMG-I蛋白的结构域 在软琼脂测定中。 将确定HMG-I/Y调节的靶基因 使用来自诱导型HMG-I/Y细胞的代表性差异分析 线 4)探索Mad、Mxi和AP-2在调节 HMG-I/Y。 额外的共转染实验和诱变分析 使用HMG-I/Y启动子构建体进行。 从这些研究中获得的见解应该会促进我们对 与c-myc和/或HMG-I/Y表达增加相关的恶性肿瘤， 可能会带来新的治疗策略。

DESCRIPTION: (adapted from the investigator's abstract) Normal cellular proliferation is initiated by growth factors that bind to their specific receptors and ultimately results in the sequential expression of genes important in regulating cell growth. Although a number of cell cycle regulatory genes have been identified, further elucidation of this genetic program will enhance our understanding of normal and neoplastic cell growth. Immediate-early genes are expressed within minutes of growth factor stimulation and include the c-myc proto-oncogene; delayed-early genes are activated hours after growth factor stimulation, but before DNA synthesis, and include the HMG-I/Y gene. To better understand the mechanisms by which delayed-early genes are activated in the G1/S transition of the cell cycle, we have been studying the regulation of the HMG-I/Y gene. Their laboratory has recently shown that HMG-I/Y is a gene target of c-Myc. Their preliminary results also indicate that the HMG-I isoform alone causes neoplastic transformation in an experimental cell line. Because expression of both c-myc and HMG-I/Y are correlated with cellular proliferation and neoplastic transformation, we hypothesize that HMG-I/Y is an important c-Myc target gene required by c-Myc for the regulation of normal and neoplastic cell growth. In addition, HMG-I/Y expression is increased in cancerous cell lines not characterized by elevated c-myc expression. They, therefore, propose that HMG-I may also lead to neoplastic transformation, independent of c-Myc. Thus, the major focus of this research proposal will be to define the role of HMG-I/Y in neoplastic transformation. The specific aims and basic experimental approaches outlined in this proposal include: 1) Evaluate the role of HMG-I/Y in transformed cell lines characterized by increased c-myc and/or HMG-I/Y expression. Antisense and potential dominant-negative HMG-I/Y vectors will be transfected into appropriate cell lines and subjected to soft agar transformation assays to address this aim. 2) Determine if HMG-I/Y plays a role in apoptosis induced by c-Myc. Antisense and potential dominant-negative HMG-I/Y vectors will also be used. In addition, we will determine if ectopic expression of HMG-I/Y leads to apoptosis. 3) Investigate the mechanisms by which HMG-I leads to neoplastic transformation. By mutagenesis analysis, we will identify the domains of the HMG-I protein required for cell transformation in soft agar assays. Target genes regulated by HMG-I/Y will be determined using representational difference analysis from an inducible HMG-I/Y cell line. 4) Explore the role of Mad, Mxi, and AP-2 in the regulation of HMG-I/Y. Additional co-transfection experiments and mutagenesis analysis using the HMG-I/Y promoter constructs will be performed. Insight gained from these studies should advance our understanding of malignancies associated with increased c-myc and or HMG-I/Y expression and may lead to new treatment strategies.