Deletion of p 16 INK 4 A / CDKN 2 and p 15 INK 4 B in Human Somatic Cell Hybrids and Hybrid-derived Tumors 1

Deletion or epigenetic inactivation of the tumor suppressor gene p16/CDKN2 (p16) has been observed in multiple human tumors. We assayed hybrid cell lines between human diploid fibroblasts and fibrosarcoma cells for p16 allelic status and expression and found that p16 was expressed in the parental diploid fibroblast cell lines used, whereas the parental fibrosarcoma cell line HT1080.6TG exhibited homozygous deletion of p16. Most immortalized hybrid cell lines derived from these parent cell lines, whether tumorigenic or nontumorigenic, exhibited loss of fibroblast-derived p16 alleles. All p16-negative hybrid cell lines also exhibited deletion of p15 (p15). Hybrid cell lines yielded tumors upon s.c. injection into athymic nude mice regardless of p16/p15 status. Tumors derived from six p16/p15-positive hybrid cells, however, revealed deletions of both p16 and p15. When human diploid fibroblasts were fused with A388.6TG squamous cell carcinoma cells, which exhibit aberrant methylation of p16, the resulting hybrids again exhibited deletion of the unmethylated fibroblastderived p16 alleles. Transfection of both HT1080.6TG and A388.6TG cells with wild-type p16 expression vector resulted in decreased clonogenicity in culture. Although the determinants directing genetic versus epigenetic inactivation of p16 and p15 remain unclear, these results demonstrate that p16-mediated growth suppression could be abrogated by either mechanism in somatic cell hybrids. Introduction Inactivation of the cyclin-dependent kinase 4 inhibitorencoding genes p16/CDKN2 (p16) and p15 (p15) in many tumors strongly suggests a role for these genes in the regulatory programs disturbed during cellular transformation. Although homozygous deletions involving chromosomal segment 9p21 in melanoma cell lines led to the initial identification of these genes (1, 2), deletions involving one or both loci have been described in multiple tumors, including cancers of the head and neck (3, 4), prostate, bladder, and lung (3); tumors of the central nervous system (5); and acute leukemias (6). Interestingly, although germ-line mutations of p16 were identified in families exhibiting a predisposition to melanoma (7), point mutations in the p16 gene in sporadic tumors appear to be relatively uncommon (8–10). More recently, epigenetic transcriptional repression, characterized by aberrant methylation of the CpG-rich promoter, has been shown to inactivate both p16 (11–15) and p15 (16) in a variety of tumors. Similar aberrant epigenetic silencing has also been shown to inactivate the retinoblastoma (17), von Hippel-Lindau (18), and human mutL homologue (19) tumor suppressor genes in nonfamilial tumors. Somatic cell hybrids constructed by fusing neoplastic and nonneoplastic cells have proven to be important tools for testing the relevance of putative tumor-inducing inactivating mutations observed in primary tumors and cell lines. The recapitulation in the hybrid cells of these deletions and mutations on alleles derived from the nonneoplastic parental cell line, associated with the acquisition of neoplastic phenotypes, provides functional evidence for tumor suppressor activity encoded by the gene or within the chromosomal region in question (20, 21). We assessed the p16 and p15 status of somatic cell hybrids constructed between nonimmortalized human fibroblasts and either human fibrosarcoma cells, in which p16 and p15 are deleted, or human squamous cell carcinoma cells, in which p16 exhibits epigenetic inactivation. The results support the genetic data, indicating a growth suppressor role for p16 and p15. The data underscore the applicability of somatic hybridization experiments to the study of epigenetic mechanisms in tumorigenesis and demonstrate the potential role of epigenetic processes in gene inactivation events associated with neoplastic progression in somatic cell hybrids.