INDUCTION OF APOPTOSIS IN THECAL INTERSTITIAL CELLS - ACTION OF TRANSFORMING-GROWTH-FACTOR-(TGF)-ALPHA PLUS TGF-BETA ON BCL-2 AND INTERLEUKIN-1-BETA-CONVERTING ENZYME/

Follicular atresia is characterized by the initial rapid loss of granu losa cells by apoptosis, followed by the loss of thecal cells at a slo wer rate. We have previously shown that treatment of subconfluent cult ures of thecal/interstitial cells (T/I) with transforming growth facto r (TGF) alpha plus TGF beta caused chromatin condensation and internuc leosomal fragmentation characteristic of apoptosis, whereas in the pre sence of either TGF alpha or TGF beta alone the cells remained healthy . In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and inte rleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quant itative reverse transcription-polymerase chain reaction (RT-PCR) assay . Bcl-2, a cell survival gene, has been implicated ill regulating the balance between cell proliferation and cell death in physiological pro cesses. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA enc oding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were a mplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatme nt of T/I cells with TGF alpha plus TGF beta significantly decreased t he levels of bcl-2 mRNA expression. TGF alpha. plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T /I cells, followed by a progressive decline to 10% of control levels a fter 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha: plus TGF beta caused a progressive in crease in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragm entation, indicative of apoptosis, occurred after 10 h of treatment wi th TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression o f bcl-2 and ICE prior to the time at which DNA fragmentation was obser ved. We propose that the gene products of bcl-2 and ICE are involved i n the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.