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 EIa‐inducible, EIIa transcription unit of adenovirus‐2 is transcribed early in infection from two start sites (+1 or EIIaE1 and ‐26 or EIIaE2), neither of which is preceded by canonical TATA box elements. Analysis of promoter deletion and linker scanning mutations for in vivo transcriptional activity after transfection into HeLa cells has indicated the existence of two overlapping promoters in the EIIaE gene. Two regions, each approximately 30 nucleotides upstream from start sites EIIaE1 and EIIaE2, function as TATA box substitutes. A sequence centered at position ‐42 (with respect to the major start site at position +1) is essential for transcription from both sites, while an element further upstream, localized between nucleotides ‐91 and ‐62, is also required for efficient EIIaE transcription, with the 3′ border being dispensable for EIIaE2 transcription. Analysis of the entire series of EIIaE mutants, co‐transfected with an EIa‐containing plasmid, revealed that no unique sequence elements in the EIIaE1 promoter region between ‐97 and +1 were responsible for the stimulation of EIIaE1 transcription by EIa. In contrast, the EIa‐mediated augmentation of EIIaE2 template activity was mainly dependent upon a sequence, the 5′‐TTAAATTT‐3′ putative TATA box substitute, located around position ‐59.

Keratin 5 (K5) mRNA and protein are shown to be expressed in normal mammary epithelial cells in culture and are absent from tumor-derived cell lines. To extend these fridings, the full complements of keratins in normal, immortalized, and tumor cells were compared. It is shown here that normal cells produce keratins K5, K6, K7, K14, and K17, whereas tumor cells produce mainly keratins K8, K18, and K19. In immortalized cells, which are preneoplastic or partially transformed, the levels of K5 mRNA and protein are lower than in normal cells, whereas the amount of K18 is increased. Thus, K5 is an important marker in the tumorigenic process, distinguishing normal from tumor cells, and decreased K5 expression correlates with tumorigenic progression. (2, 4-11, 35, 36). Initial studies with keratins extracted from mammary glands showed differences between normal and tumor tissues (2).Immunohistochemical characterization has demonstrated that within the normal duct, basal and lumenal cells can be discriminated by the keratins they express. In these studies, basal cells, lying between the lumenal cells and the basement membrane, were characterized by expression of keratins K5 and K14, which are typical of myoepithelial cells in stratified epithelium, and lumenal cells were characterized by expression of simple epithelial keratins K8, K18, and K19 (12-15).These studies were performed in situ. Keratin expression may not be maintained after disruption of tissue architecture in cell culture. Medium constituents such as vitamin A, cAMP-elevating agents, epidermal growth factor, and other factors are known to affect keratin production (13,(16)(17)(18)37). The development of a medium capable of supporting the growth ofboth tumor and normal breast epithelial cells (1) has allowed us to make a comparison of their keratin profiles independent of medium effects. We have analyzed K5 as a potential marker for normal cells and have reviewed the array of keratins produced by cultured tumor and normal cells. cDNA Library Production. Total RNA was isolated from 184 cells by lysis with guanidinium isothiocyanate, centrifugation over a 5.7 M CsCl cushion, and purification of the resulting RNA pellet (24). Poly(A)+ RNA was purified by oligo(dT) chromatography using standard protocols (ref. 25, pp. 197-198). cDNA was made using the reverse transcriptase of Moloney murine leukemia virus (BRL) according to the supplier's instructions. cDNAs were blunt ended with T4 DNA polymerase, EcoRI linkers were ligated onto the cDNA, and the cDNA was cloned in the EcoRI site of AgtlO. MATERIALS AND METHODSSubtraction and Screening. The first-strand cDNA of 184 mRNA was hybridized with a 5-fold mass excess of 184B5KSVTu2 poly(A)+ RNA to Rot = 6000 mol (of nucleotide)-sec. Nonhybridizing nucleic acid was separated from DNARNA hybrids by chromatography over hydroxylapatite (26) and subjected to a further round of hybridization and hydroxylapatite chromatography. The final product (the "subtracted probe") was labeled by the random primer method (27)...

NGS assessment of therapy resistant OC identifies an unexpectedly high frequency of GA that could influence targeted therapy selection for the disease.

Although liver is an estrogen target tissue, the number of hepatic genes known to be directly induced by estrogen is very small. We identified proteinase inhibitor 9, or PI-9, as being rapidly and strongly induced by estrogen in an estrogen receptor-positive human liver cell line (HepG2-ER7). Since PI-9 mRNA was also induced by estrogen in a human liver biopsy sample, PI-9 is a genuine estrogen-regulated human gene. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis. Estrogens induced PI-9 mRNA within 2 h, PI-9 mRNA levels reached a plateau of 30 -40-fold induction in 4 h, and induction was not blocked by cycloheximide, indicating that induction of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamoxifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrogen ICI 182,780 was a pure antagonist. Western blot analysis showed that estrogen strongly increases PI-9 protein levels. Inhibition of transcription with actinomycin D resulted in identical rates of PI-9 mRNA decay in the presence and absence of estrogen. We isolated genomic clones containing the PI-9 promoter region, identified a putative transcription start site, and carried out transient transfections of PI-9-luciferase reporter gene constructs. The estrogen, moxestrol, elicited a robust induction from the PI-9-luciferase reporter. Mutational inactivation of three potential imperfect estrogen response elements in the PI-9 5-flanking region had no effect on moxestrol estrogen receptor induction.