Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing in a minimally supplemented dermed medium (D3) containing epidermal growth factor. All transfectants tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grow and investigate may also be immortalized by HPV.Breast cancer is a very frequent lethal malignancy of women in North America and western Europe, but the underlying molecular genetics and pathobiology are poorly understood. A major deterrent to research has been the lack of suitable cell culture systems in which primary tumor cells could be grown and compared with the normal mammary epithelial cells of origin and in which the stepwise process of mammary tumorigenesis could be investigated (1). The recent development of a medium, DFCI-1, in our laboratory should alleviate this problem (2); newly isolated cell lines from primary tumors are now being studied (ref.

The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cells, increased transgene capacity, complete replication incompetence, and elimination of replication-competent virus that can be produced during the growth of first-generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functional complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was constructed by transforming 293 cells with an inducible E4-ORF6 expression cassette. These 293-ORF6 cell lines were used to construct and propagate viruses with E1 and E4 deleted. While the construction and propagation of AdRSV␤gal.11 (an E1 ؊ /E4 ؊ vector engineered to contain a deletion of the entire E4 coding region) were possible in 293-ORF6 cells, the yield of purified virus was depressed approximately 30-fold compared with that of E1 ؊ vectors. The debilitation in AdRSV␤gal.11 vector growth was found to correlate with reduced fiber protein and mRNA accumulation. AdCFTR.11A, a modified E1 ؊ /E4 ؊ vector with a spacer sequence placed between late region 5 and the right inverted terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1 ؊ vectors. Moreover, purified AdCFTR.11A yields were equivalent to E1 ؊ vector levels. Since no overlapping sequences exist in the E4 regions of E1 ؊ /E4 ؊ vectors and 293-ORF6 cell lines, replication-competent virus cannot be generated by homologous recombination. In addition, these second-generation E1 ؊ /E4 ؊ vectors have increased transgene capacity and have been rendered virus replication incompetent outside of the new complementing cell lines.

We have shown previously that introduction of the human papillomavirus type 16 (HPV16) or HPV18 genome into human mammary epithelial cells induces their immortalization. These immortalized cells have reduced growth factor requirements. We report here that transfection with a single HPV16 gene E6 is sufficient to immortalize these cells and reduce their growth factor requirements. The RB protein is normal in these cells, but the p53 protein is sharply reduced, as shown by immunoprecipitation with anti-p53 antibody (pAB 421). We infer that the E6 protein reduces the p53 protein perhaps by signalling its destruction by the ubiquitin system. The HPV-transforming gene E7 was unable to immortalize human mammary epithelial cells. Thus, cell-specific factors may determine which viral oncogene plays a major role in oncogenesis. Human papillomavirus types 16 and 18 (HPV16 and HPV18) have been associated with cervical carcinoma, although most of the 65 or more known HPV types are restricted to small benign epidermal lesions, such as warts and papillomas (14). New HPV types and new associations with oral as well as genital carcinomas are being reported (14), but no evidence for an association of HPV with breast cancer has yet been found. Indeed, it has been postulated that the narrow host range of HPV, apparently limited to squamous epithelial cells, may reflect a specific cellular interaction provided by these cells.

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.