Immortalization of fibroblasts from two patients with hereditary retinoblastoma

Two RB fibroblastic strains from patients with hereditary retinoblastoma (RB), namely, RB80F/250R and RB110F, were transfected with plasmid DNAs encoding SV-40 large T-antigen at passage 31 and 10, respectively, These transfected fibroblasts developed into two immortalized cell lines. RB80F/250R/ori- and RB110F/gpt. RB110F/gpt had a similar doubling time as the parental cells, whereas RB80F/250R/ori- grew more rapidly with a doubling time of 19 hours compared to the parental cells which had a doubling time of 50 hours. The RB80F/250R/ori- cell line was particularly interesting cytogenetically since no normal chromosome 13 was present, although a marker chromosome 13 was found. In contrast, two apparently normal chromosome 13s were present in RB110F/gpt cells, but these cells had a marker chromosome which involved chromosome 14 (14 p+). Both the cell lines also had an abnormal chromosome 1 (1q-). RFLP analysis using the chromosome 13 specific VNTR probe, pTH162, assigned to 13q14.1 showed that the DNA from the RB80F/250R/ori- cells contained only a 6.1kb allelic fragment whereas the DNAs from parental RB80F/250R and RB80F cultures demonstrated both the polymorphic 8.0kb and 6.1kb allelic fragments. However, the RB gene per se was normal at the DNA level. Both cell lines expressed SV-40 large T-antigen together with elevated levels of p53 protein. In addition, levels of RB protein were the same in exponentially growing nontransformed parental cell strains and their immortalized cell lines. However, at confluency the levels of RB protein were greatly reduced in nontransformed cells but not in immortalized cell lines under similar conditions. In future studies using these immortalized cell lines, we shall make an attempt to discern the role of the RB gene and other tumor suppressor genes in the regulation of normal and malignant growth.