Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13

Isolation of cell lines that display various degrees of transformed phenoty pes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from h uman diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RBI gene is located. By transfection with SV40 early genes and repeate d passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting s howed that these cell lines were not contaminants, but derivatives of the o riginal RB cells. The remaining RBI allele may be wild-type even in the mal ignant cell lines, because the expression and the LT-binding ability were n ormal. Furthermore, we did not find any homozygous loss in 16 polymorphic m arkers located in the 13q14-22 region in the transformed cell lines. Howeve r, introduction of a copy of a normal chromosome 13 into the anchorage-inde pendent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletio n progressed to a tumorigenic state spontaneously, but normal fibroblasts d id not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclud e that these RB cell lines provide an excellent system for identification o f genes involved in malignant transformation of human cells. (C) 1999 Wiley -Liss, Inc.