Molecular and genetic characterization of human keratinocytes transformed with ionizing radiation

Abstract

SUMMARY The molecular mechanisms which underlie the biological effects of exposure to ionizing radiation have particular relevance to radiation protection and risk assessment since most cancers are of epithelial origin. It is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Our previous studies have demonstrated that both X-rays and fission neutrons can transform immortalized human epidermal keratinocytes to a malignant phenotype. In both studies, exposure to ionizing radiation led to the development of morphologically altered cells and focus formation at confluence. These radiation-transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth, and formed tumors in athymic mice. These radiation studies have been expanded to include a molecular analysis of the cellular ras genes and the tumor suppressor gene p53. Single-strand conformational polymorphism (SSCP) analysis and DNA sequencing demonstrated the absence of point mutations, deletions, and rearrangements in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 2-11 of the p53 tumor suppressor gene. Karyotypic analysis of these human keratinocytes indicated a loss of heterozygosity at the tumor suppressor loci RB1, DCC, APC, MTS1, and WT1 upon immortalization. Furthermore, karyotypic analysis of the X-ray-treated keratinocytes in relation to their tumorigenic potential indicated that the development of malignancy was associated with a duplication of material on chromosome 11 between regions q14 and q22. This additional material on chromosome 11 a t q14/q22 was observed in all of the irradiated tumorigenic keratinocytes. A similar tandem duplication of the llq13>q23 region has been observed in malignant lesions of oral squamous cell carcinoma and in head and neck tumors. Deletions in the same region were also observed in the irradiated non-tumorigenic cells. Together, these observations indicate that this region of chromosome 11 contains gene@) with a role in tumorigenicity and that the duplication observed in chromosome 11 may be the primary event leading to radiationinduced transformation of these immortalized human keratinocytes. These studies demonstrate that mutations in either the cellular pS3 or rus genes do not occur in these immortalized human epithelial cells (RHEK-1) when transformed to a malignant phenotype with ionizing radiation. Furthermore, specific regions on chromosome 11 may be a target for the transforming effects of ionizing radiation.