Effects of simian virus 40 T-antigens on normal human mammary epithelial cells reveal evidence for spontaneous alterations in addition to loss of p16(INK4a) expression.

Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression.

Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.

Telomerase-negative immortalized human cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (ALT). We report here that ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB). APBs are large donut-shaped nuclear structures containing PML protein, telomeric DNA, and the telomere binding proteins human telomere repeat binding factors 1 and 2. Immunostaining showed that APBs also contain replication factor A, RAD51, and RAD52, proteins involved in DNA synthesis and recombination. During immortalization, APBs appeared at exactly the same time as activation of ALT. APBs were found in ALT tumors and cell lines but not in mortal cell strains or in telomerase-positive cell lines or tumors.

Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells.

Inactivation of p16INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M0). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16INK4 expression in postselection HMECs. In contrast, p16INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16INK4 CpG island and loss of p16INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

Association of extended in vitro proliferative potential with loss of p16INK4 expression.

This study addresses the question of whether loss of p16INK4 expression contributes to the immortalization of human cells. In vitro immortalization usually proceeds through two phases. In the first phase (lifespan extension), cells continue proliferating and their telomeres continue shortening beyond the point at which normal cells become senescent. In the second phase (immortalization), the cells activate a telomere maintenance mechanism and acquire an unlimited proliferative potential. It has previously been shown that immortalized cells containing viral oncoproteins that bind and inactivate p110RB contain wild-type p16INK4; we therefore examined the p16INK4 status of cell lines that became immortalized in vitro in the absence of these oncoproteins. Three such lines were identified: III-CF/.2A1 and III-CF/E6A2 (both derived from Li-Fraumeni syndrome fibroblasts, probably by spontaneous immortalization) and MePV-231 (normal mesothelial cells transfected with HPV-16 E6/E7 genes that underwent deletion of these genes before immortalization). In each case p16INK4 expression was lost at or before immortalization. Further, a cell strain was identified that had an extended but finite lifespan associated with loss of p16INK4 (and p53) expression. Thus loss of p16INK4 expression was associated with extended in vitro lifespan but was not sufficient for immortalization, even in the absence of wild-type p53.

A novel human cDNA highly homologous to the fish hormone stanniocalcin.

Stanniocalcin is a glycoprotein hormone previously considered present only in bony fish where it is secreted by the corpuscles of Stannius, endocrine organs involved in Ca2+ homeostasis. In fish, stanniocalcin was thought to be an adaptation for Ca2+ regulation in aquatic environments, and its effects include inhibition of gill Ca2+ transport. We have obtained a human cDNA clone coding for a protein highly homologous to fish stanniocalcin. The mRNA is expressed in many human tissues, with the highest levels in ovary, prostate and thyroid. In vitro human cell culture studies show that the mRNA is positively regulated by extracellular Ca2+ in the medium. We conclude that a human protein similar to the fish hormone is expressed in multiple tissues rather than by a specialized endocrine organ.

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53.

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.

Maxi-pouch: a new technique for ileal conduit conversion to continent urinary reservoir.

We present an alternative technique for conversion of an ileal conduit to a continent urinary diversion that uses a combination of ileal and ileocolonic substitutions. Filling pressure studies and radiography of the pouch, endoscopy and excretory urography revealed a reservoir capacity of 850 cc, no reflux, normal urinary tracts and no uninhibited contractions. In complex cases of neuropathic and nonneuropathic bladders or patients who have undergone cystectomy and have an unacceptable lower urinary tract for undiversion, we propose this technique of continent urinary reservoir as an alternative.

Role of keratinocytes in human recurrent herpetic lesions. Ability to present herpes simplex virus antigen and act as targets for T lymphocyte cytotoxicity in vitro.

In human recurrent herpetic lesions epidermal keratinocytes are induced to express HLA class II (DR) antigens. Keratinocytes derived from human split skin and cultured in vitro were induced to express HLA-DR but not -DQ antigens with IFN gamma preparations. These stimulated keratinocytes presented herpes simplex antigen directly to autologous blood-derived T lymphocytes in four of four subjects (stimulation indices: 1.5-2.7), suggesting that keratinocytes may have an accessory herpes simplex virus (HSV) antigen-presenting role in addition to the Langerhans cells and macrophages in herpetic skin lesions. Blood mononuclear cells from eight herpes simplex seropositive subjects which were activated in vitro by HSV antigen for 6 d showed cytotoxicity specific for HSV in infected autologous keratinocytes. This was significantly increased by prestimulation with IFN gamma (51-56% to 83-85%). In four of eight patients some cytotoxicity also occurred against uninfected, IFN gamma-stimulated keratinocytes. Lymphocyte subset analysis showed that cytotoxicity against HSV-infected, IFN gamma-stimulated keratinocyte targets was mediated by both CD3+ T lymphocytes and Leu 11b+ natural killer cells. T lymphocyte cytotoxicity was mediated by both CD4+ and CD8+ T lymphocytes, suggesting a cytotoxic role for the activated CD4+ lymphocytes that initially predominate in herpetic lesions.