FUNCTIONAL EXPRESSION OF SV40 IN NORMAL HUMAN PROSTATIC EPITHELIAL AND FIBROBLASTIC CELLS - DIFFERENTIATION PATTERN OF NONTUMORIGENIC CELL-LINES

To study mesenchymal-epithelial interactions associated with the normal and pathological human prostate, we have developed a model of well differentiated human prostate epithelial and fibroblastic cells. Normal human prostatic cells, either of epithelial or fibroblastic origins were successfully transfected with SV40 and strains with extended lifespan were selected until the crisis was reached, within 20 and 30 passages for the epithelial and fibroblastic cells, respectively. Only a few clones emerged from the crisis: PNT1A (Cussenot et al: J Urol 143: 881-886, 1991), PNT1B and PNT2 epithelial cell lines. Successful immortalisation was achieved only with SV40 expressing both large T and small t oncogenes, while attempts to immortalise with a vector expressing SV40 large T alone have given a few strains showing no extended lifespan and no cells which overcame the crisis. A PNT2 subclone named PNT2-LSD which developed spontaneously (less serum dependent) was selected, characterised and included in the analysed series. The epithelial cell lines displayed a differentiation pattern which has been classified as follows (from high to low): PNT2>PNT2-LSD>PNT1A>PNT1B. Differentiation features studied were (i) the colony-forming ability of the PNT2 and PNT2-LSD compared to PNT1A and PNT1B, (ii) their respective doubling time of 39, 29, 30 and 28 hours, (iii) their decreasing serum dependency, (iv) the expression of cytokeratin 19 (a feature of well differentiated luminal cells of the glandular prostate) for PNT2 and PNT2-LSD. Furthermore, the mesenchymal derived pflsv1 cells were confirmed to be of fibroblastic nature. None of the cell lines analysed showed any tumourigenicity in nude mice over a period of 12 months. Serum deprivation and direct steroid withdrawal during the culture triggered cell death by apoptosis, an event which could be overcome by EGF stimulation, particularly for the well differentiated PNT2 cells. This interesting characteristic, which is similar to the high apoptotic rate observed ipl vivo for normal prostate, particularly after castration should lead, together with the other properties of these cell lines, to a better understanding of the biology of the different cell compartments involved in the progression of prostate towards neoplasia.