EFFECT OF 3 TUMOR PROMOTERS ON THE STABILITY OF HEPATOCYTE CULTURES AND APOPTOSIS AFTER TRANSFORMING GROWTH-FACTOR-BETA-1

Tumour promoters like the anti-androgen cyproterone acetate (CPA), the peroxisome proliferator nafenopin (NAF) and phenobarbital (PB) stimul ate liver growth in rodents. Transforming growth factor-beta 1 (TGF-be ta 1) is expressed in livers after treatment with CPA (Oberhammer et a l., submitted) and some peroxisome proliferators. In this paper we des cribe the influence of CPA, NAF and PB on the stability of hepatocyte cultures and induction of apoptosis by TGF-beta 1. All three tumour pr omoters had a stabilizing effect on confluent monolayers of hepatocyte s, partially preventing the usually occurring dedifferentiation and de tachment processes. CPA on its own was able to induce apoptosis at the high dose of 10 mu M No induction of apoptosis could be observed afte r PB and NAF. At any dose above 0.01 mu M CPA enhanced TGF-beta 1-indu ced apoptosis (5.8-fold increase with 10 mu M CPA). Thus the combinati on of 10 mu M CPA and 1 ng/ml TGF-beta 1 induced apoptosis in 90% of t he plated hepatocytes. At a high dose (10 mu M) NAF produced a 35% red uction in apoptosis induced by TGF-beta 1, in parallel with a stabiliz ing effect on cell number. PB did not affect the rate of apoptosis ind uced by TGF-beta 1. As demonstrated by immunohistochemical detection o f PCNA, TGF-beta 1 prevented induction of PCNA by epidermal growth fac tor (EGF). No induction of PCNA was observable in CPA-treated cultures . In untreated and EGF-treated cultures TGF-beta 1 was able to induce apoptosis to the same extent within 30 h. In CPA-treated cultures this period was shortened to 12 h. Thus CPA shortens the lag phase of indu ction of apoptosis by shifting hepatocytes to a point before S phase, where they are highly susceptible to TGF-beta 1-induced apoptosis.