EXPERIMENTAL CELL RESEARCH 235, 245–253 (1997) ARTICLE NO. EX973670 Genomic Instability and Telomerase Activity in Human Bronchial Epithelial Cells during Immortalization by Human Papillomavirus-16 E6 and E7 Genes Jill D. Coursen,* ,1 William P. Bennett, * ,1 Lauren Gollahon,† Jerry W. Shay,† and Curtis C. Harris* ,2 *Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255; and †Department of Cell Biology and Neuroscience, UT Southwestern Medical Center, Dallas, Texas 75235-9039 INTRODUCTION Human papilloma virus types 16 and 18 contribute The papillomaviruses are small, nonenveloped DNA to the development of cervical carcinomas in which viruses. The human papilloma viruses (HPV) are di- the E6 and E7 genes are frequently retained and ex- vided into low-risk and high-risk types based on their pressed in the tumors. Our study explored the ability association with human cancers. Low-risk HPV types, of the E6 and/or E7 genes to immortalize normal hu- including types 6 and 11, result in benign lesions such man bronchial epithelial (NHBE) cells and to reacti- vate telomerase expression in these cells. We have in- as genital warts. HPV types 16 and 18 are considered troduced the human papillomavirus type 16 E6 or E7 high risk because they are associated with the majority genes alone or in combination (E6/E7) into NHBE cells of invasive cervical carcinomas (reviewed in [1]). The using the retroviral construct pLXSN. Cells expressing human papilloma viruses specifically infect epithelial either the E6 or the E7 oncoproteins alone displayed cells. This tissue trophism may be determined by an an increased colony-forming efficiency and a slightly epithelial-specific transcription enhancer, which was extended in vitro life span before entering a crisis, first identified in HPV-16 and -18 (reviewed in [2]). from which immortalized cell lines were not obtained. HPV DNA sequences have been detected in 84% of hu- Telomerase activity was not detected in cells express- man cervical cancers [3 – 5] and less frequently in respi- ing either E6 or E7 individually. Cells expressing the ratory tract cancers [6 – 9]. This transforming ability of E6/E7 oncoproteins in combination had a substantially HPV-16 and -18 has been attributed to the E6 and E7 increased life span before entering crisis. A subpopula- proteins, which are frequently retained and expressed tion of these cells escaped from crisis and achieved in tumors. 130 population doublings, suggesting immortalization. HPV-16 E6 and E7 act by binding to cellular pro- Telomerase activity was detected in these postcrisis teins. The E6 protein binds to the p53 tumor-suppres- cells, but was not detected prior to crisis. In addition, sor protein, resulting in enhanced ubiquitin degrada- karyotypic analysis showed evidence of genomic insta- tion of p53 [10, 11]. This interaction between E6 and bility in mass cultures as well as clones expressing E6, p53 is mediated by the cellular protein E6-AP, which E7, or E6/E7. Abnormalities included numerous mono- is a component of the ubiquitin pathway [12–14]. It somies and trisomies, chromatid gaps and breaks, dou- has been demonstrated in human mammary epithelial ble minutes, and aberrant chromosomes. These results cells that degradation of p53 is required for their im- demonstrate that expression of E6 and/or E7 is suffi- mortalization by E6 [15]. p53 is frequently mutated in cient to induce genomic instability and an extended life span to NHBE cells, but the presence of both E6 a variety of human cancers [16] and it is currently and E7, along with at least one additional genetic or thought that its main function is to induce cellular epigenetic event achieved during crisis, was required growth arrest and/or apoptosis in response to DNA for reactivation of telomerase and the immortalization damage [17]. In addition, p53 is important for main- in this human cell type.  1997 Academic Press taining the fidelity of centrosomal duplication and lack of wild-type p53 leads to aneuploidy [18]. Therefore, inactivation of p53 by E6 could permit aneuploidy and the accumulation of genetic damage which could lead 1 These authors have contributed equally to this study. to neoplastic transformation. In fact, it has been dem- 2 To whom correspondence and reprint requests should be ad- onstrated that E6, as well as E7, can inhibit DNA dam- dressed at Laboratory of Human Carcinogenesis, National Cancer age-induced growth arrest [19, 20] and produce aneu- Institute, Building 37, Room 2C05, 37 Convent Drive MSC ploidy [21, 22]. E6 also can abrogate p53-mediated 4255, Bethesda, MD 20892-4255. Fax: (301) 496-0497. E-mail: Curtis_Harris@nih.gov. DNA binding [23, 24], transcriptional transactivation 245 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.