Abstract: 406

BASIC FIBROBLAST GROWTH FACTOR (bFGF) INHIBITS THE APOPTOTIC DEATH OF SPONTANEOUSLY IMMORTALIZED GRANULOSA CELLS (SIGCs) THROUGH A PROTEIN KINASE C-DEPENDENT PATHWAY.

Kelly Lynch¹ , John Peluso^{1 *}
Departments of Physiology and Obstetrics and Gynecology, University of CT Hlth Ctr, Farmington, CT. ¹

Previous studies has shown that bFGF inhibits primary granulosa cells from undergoing apoptosis but the signal transduction pathway through which bFGF mediates its anti-apoptotic action has not been clearly identified. The present study was designed to determined whether bFGF mediates its action through a protein kinase C (PKC)-dependent mechanism. SIGCs were cultured in serum-supplemented medium for 24 h, then in serum-free medium in the presence or absence of bFGF (10 ng/ml). After 5 h, apoptosis was assessed in situ by staining the cells with YOPRO-1. In the absence of serum, the percentage of apoptotic cells increased from 9 ± 1% to 24 ± 1% (p < 0.05). This increase was reduced to 12 ± 1% by bFGF (p < 0.05). If the addition of bFGF was delayed for 30 but not 15 mins after the removal of serum, bFGF failed to inhibit apoptosis. Further, an activator of PKC (the phorbol ester TPA at 10 or 100 nM) blocked apoptosis due to serum deprivation. Like bFGF, TPA had to be present between 15 and 30 min after serum withdrawal in order for TPA to prevent apoptosis. Conversely, an inhibitor of PKC (Bisindolylmaleimide II) completely attenuated, while Bisindolylmaleimide V ( a negative control for PKC inhibitors) did not influence bFGF’s anti-apoptotic action. These observations suggest that bFGF activates PKC within the first 30 min of serum withdrawal. If PKC is not activated during this time, then a critical event occurs that results in an increase in apoptosis by 5 h of culture. Since BAPTA (5 M), an intracellular chelator of calcium, prevents SIGCs from undergoing apoptosis in serum-free medium, it is possible that PKC activation is involved in maintaining calcium homoeostasis. (Supported by NIH Grant HD 33467).

    This abstract is being presented on Monday, August 2 at 2:15 PM at Todd 125.