RETINOIDS & PROTEIN KINASE C IN MELANOMA DIFFERENTIATION

维生素A的主要生理活性代谢物--维甲酸 (RA)，在预防某些类型的癌症方面很重要。RA也可以 抑制多种肿瘤细胞的生长并诱导其分化 在文化中，并被用来治疗某些癌症。我们一直在研究 RA诱导黑色素瘤分化。一个重要的发现是 PKCalpha在分化途径中起关键作用。我们会 继续研究PKC和PKC介导的事件在 黑色素瘤分化。具体来说，我们将确定RARbeta是否 直接参与了PKCalpha的诱导和随后的 分化方式：建立稳定的转染体，其中 RARbeta的表达受诱导型启动子的控制； 通过同源重组破坏RARbeta基因；使用新的 维甲酸类似物，具有激活RARbeta的特异性。通过 结合来自三种不同方法的数据，我们应该获得 关于RARbeta在黑色素瘤中作用的令人信服的证据 差异化。我们发现RA也能诱导AP-1的增加 活动。这不是因为c-fox或c-jun的增加，也不是因为 与AP-1元件的结合增加，但显然是由于 C-jun氨基末端激酶(JNK)的转录活性，或 增加它的表达。为了确定AP-1的重要性 分化活性我们将分离出稳定的转染体 表达c-jun的显性负性突变体，并检测这些克隆 对类风湿关节炎是难治性的。我们还将使用新开发的维甲酸类似物 它们区分了RAR的反式激活和AP-1反式抑制。 上一次赠款期间的一个令人兴奋的发现是，PKC可以 正向调节RAR功能，从而形成正反馈循环。 我们将研究PKCalpha是否可以直接磷酸化RARs 以及这种磷酸化如何影响受体功能。这些数据将 提供对RA介导的肿瘤细胞过程的进一步洞察 差异化，并可以提出方法，通过这个过程 增强了。

The major physiologically active metabolite of vitamin A, retinoic acid (RA), is important in preventing certain types of cancer. RA can also arrest the growth and induce differentiation of a variety of tumor cells in culture and is used to treat certain cancers. We have been studying RA-induced melanoma differentiation. An important finding has been the key role played by PKCalpha in the differentiation pathway. We will continue studies on the involvement of PKC and PKC-mediated events in melanoma differentiation. Specifically, we will determine whether RARbeta is directly involved in the induction of PKCalpha and subsequent differentiation by: establishing stable transfectants in which the expression of RARbeta is under the control of an inducible promoter; disrupting the RARbeta gene through homologous recombination; use of new retinoid analogs which have specificity for activating RARbeta. By combining the data from three separate approaches we should obtain convincing evidence regarding the role of RARbeta in melanoma differentiation. We found that the RA also induces an increase in AP-1 activity. This is not due to an increase in c-fox or c-jun nor to an increase in binding to an AP-1 element, but apparently to an increase in transcriptional activity of c-jun amino-terminal kinase (JNK), or increasing its expression. In order to determine the importance of AP-1 activity for differentiation we will isolate stable transfectants which express a dominant-negative mutant of c-jun and test whether these clones are refractory to RA. We will also use newly developed retinoid analogs which distinguish between transactivation and AP-1 transrepression of RAR. An exciting finding of the previous grant period was that PKC could positively regulate RAR function, thus creating a positive feed-back loop. We will investigate whether PKCalpha can directly phosphorylate the RARs and how this phosphorylation affects receptor functions. These data will provide further insight into the process of RA-mediated tumor cell differentiation and could suggest means by which the process could be potentiated.