Loss of Suppressor Loci on Chromosomes 11 and 14 May Be Required for Radiation-Induced Neoplastic Transformation of HeLa × Skin Fibroblast Human Cell Hybrids

Abstract

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Monochromosome transfer provides functional evidence for growth‐suppressive genes on chromosome 14 in nasopharyngeal carcinoma

“…Previous findings imply that loss of the entire chromosome 14 is associated with tumorigenicity of certain tumor hybrids (Srivatsan et al, 1986;Mendonca et al, 1998Mendonca et al, , 2000, but the location of candidate genes on this chromosome is presently not clear. In recent years, genetic losses of chromosome 14 have often been reported from different human tumors, including NPC.…”

Homozygous deletions within the 11q13 cervical cancer tumor‐suppressor locus in radiation‐induced, neoplastically transformed human hybrid cells

“…However, after a standard dose of 7 Gy of gamma or X-rays, radiation-induced transformation frequency of 4 -8 ϫ 10 Ϫ4 (40 -80 times above the spontaneous transformation frequency) could be observed (Redpath et al, 1987;Sun et al, 1988;Mendonca et al, 1990Mendonca et al, , 1998b. Molecular genetic characterization of these radiation-induced, neoplastically transformed cell lines (gamma-induced mutants, or GIMs) also has shown that the reversion to a tumorigenic phenotype was strongly associated with the loss of a fibroblast chromosome 11 (Mendonca and Redpath, 1989;Mendonca et al, 1991Mendonca et al, , 1995Mendonca et al, , 1998a. These hybrids had lost a copy of fibroblast chromosome 11 while retaining an intact copy of fibroblast chromosome 11.…”

“…Ten micrograms of the genomic DNA from each cell line was digested with various restriction enzymes. The digested DNAs were separated on 0.8% agarose gels (SeaKem, FMC Bioproducts, Rockland, ME) and transferred onto nylon membranes and hybridized to random primed 32 P-labeled probes (Feinberg and Vogelstein, 1983) as described previously (Mendonca et al 1995(Mendonca et al , 1998a. Because of the presence of repeat DNA in the probe sequences, preannealing at 65°C with Cot-1 DNA was required to reduce background.…”

“…Because of the presence of repeat DNA in the probe sequences, preannealing at 65°C with Cot-1 DNA was required to reduce background. Hybridization and membrane washing were done according to a standard protocol (Mendonca et al, 1995(Mendonca et al, , 1998a. The blots were wrapped in Saran wrap and exposed to Kodak X-OMAT AR film at Ϫ70°C for 1-6 days with an intensifying screen.…”

“…High-molecular-weight DNA was isolated by standard techniques from the various cell lines (Mendonca et al, 1995(Mendonca et al, , 1998a. Primers for chromosome band 11q13 were identified from 11q13-specific cosmid and BAC sequences, and all other primers were selected from the chromosome 11 database.…”