RETINOIDS & PROTEIN KINASE C IN MELANOMA DIFFERENTIATION

维生素A的主要生理活性代谢产物，视黄酸 (RA)，对预防某些类型的癌症很重要。 RA也可以 抑制多种肿瘤细胞的生长并诱导其分化 并用于治疗某些癌症。 我们一直在研究 RA诱导的黑素瘤分化。 一个重要的发现是， PKCalpha在分化途径中发挥的关键作用。 我们将 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PKCalpha的诱导和随后的 通过以下方式分化：建立稳定的转染子， RAR β的表达在诱导型启动子的控制下; 通过同源重组破坏RAR β基因;使用新的 对激活RAR β具有特异性的类维生素A类似物。 通过 结合三种不同方法的数据， 关于RAR β在黑色素瘤中作用的令人信服的证据 分化 我们发现，RA还诱导AP-1的增加， 活动 这不是由于c-fox或c-jun的增加，也不是由于 与AP-1元件结合的增加，但显然与 c-jun氨基末端激酶（JNK）的转录活性，或 增加其表达。 为了确定AP-1 我们将分离稳定的转染子， 表达c-jun的显性阴性突变体，并测试这些克隆是否 难治性RA。 我们还将使用新开发的维甲酸类似物 其区分RAR的反式激活和AP-1反式阻遏。 上一个资助期的一个令人兴奋的发现是，PKC可以 积极调节RAR功能，从而创建一个正反馈循环。 我们将研究PKCalpha是否可以直接磷酸化RAR 以及这种磷酸化作用如何影响受体功能。 这些数据将 进一步了解RA介导的肿瘤细胞 差异，并可能建议该过程可以 增强

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.