LIVER-CELLS CAN SPONTANEOUSLY RESUME PROLIFERATION IN LONG-TERM QUIESCENT PRIMARY CULTURES

We report here data on the spontaneous resumption of proliferation in long-term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum-supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying gamma-glutamyl-transpeptidase (gamma-GT) activity and F-actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15-week-old primary cultures were highly tumorigenic, with a 3-6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin-enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hyperploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3 gamma-GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. alpha2 macroglobulin, PMSF) and 5 tumorigenicity. Long-term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.