Effect of three tumour 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) stimulate liver growth in rodents. Transforming growthfactor-ß1(TGF-ß1) is expressed in liversafter treatment with CPA (Oberhammer et at., submitted) andsome peroxisome proliferators. In this paper we describe theinfluence of CPA, NAF and PB on the stability of hepatocytecultures and induction of apoptosis by TGF-ß1. Allthree tumour promoters had a stabilizing effect on confluentmonolayers of hepatocytes, partially preventing the usuallyoccurring dedifferentiation and detachment processes. CPA onits own was able to induce apoptosis at the high dose of 10µM. No induction of apoptosis could be observed afterPB and NAF. At any dose above 0.01 µM CPA enhanced TGF-ß1-inducedapoptosis (5.8-fold increase with 10 µM CPA). Thus thecombination of 10 µM CPA and 1 ng/ml TGF-ß1induced apoptosis in 90% of the plated hepatocytes. At a highdose (10 µM) NAF produced a 35% reduction in apoptosisinduced by TGF-ß1 in parallel with a stabilizing effecton cell number. PB did not affect the rate of apoptosis inducedby TGF-ß1. As demonstrated by immunohistochemicaldetection of PCNA, TGF-ß1 prevented induction of PCNAby epidermal growth factor (EGF). No induction of PCNA was observablein CPA-treated cultures. In untreated and EGF-treated culturesTGF-ß1 was able to induce apoptosis to the same extentwithin 30 h. In CPA-treated cultures this period was shortenedto 12 h. Thus CPA shortens the lag phase of induction of apoptosisby shifting hepatocytes to a point before S phase, where theyare highly susceptible to TGF-ß1-induced apoptosis.