2-METHOXYESTRADIOL-INDUCED PHOSPHORYLATION OF BCL-2 - UNCOUPLING FROMJNK SAPK ACTIVATION/

The natural estrogen metabolite 2-methoxyestradiol (2ME) is anti-angio genic in vivo and a strong growth inhibitor in vitro. The growth inhib ition is due to mitotic arrest and apoptosis. These effects are remini scent of those induced by taxol, and appear to be mediated by inhibiti on of microtubule dynamics. Here we have studied the cellular response to 2ME in regard to potential mediators of the observed cellular chan ges. 2ME treatment increases the insoluble polymerized fraction of cel lular tubulin similar to taxol, and in contrast to the microtubule dep olymerizing drugs such as colcemid and vincristine. This stabilization following 2ME treatment is accompanied by phosphorylation and inactiv ation of Bcl-2 increasing gradually hom 2-24 hours. To study the pathw ay leading to Bcl-2 phosphorylation we analyzed Raf-1 and JNK/SAPK kin ases, both of which have been reported to be involved in Bcl-2 inactiv ation. Our results indicate that Raf-1 is phosphorylated in response t o 2ME, but this occurs later than Bcl-2 phosphorylation suggesting tha t Raf-1 is not directly phosphorylating Bcl-2. JNK/SAPK was activated rapidly after 2ME treatment. However, this activation was transient an d returned to undetectable levels by 2 hours of treatment, demonstrati ng that JNK/SAPK is not directly phosphorylating Bcl-2. Taken together with previous results indicating that overexpression of JNK/SAPK lead s to Bcl-2 phosphorylation, our results would support a model where JN K/SAPK is indirectly phosphorylating Bcl-2. (C) 1998 Academic Press.