A nine-codon deletion mutation in the cytomegalovirus UL97 phosphotransferase gene confers resistance to ganciclovir.

A deletion mutation (codons 595 to 603) in the cytomegalovirus (CMV) UL97 gene was recently reported after sequence analysis of leukocyte DNA from a patient receiving ganciclovir. The corresponding viral phenotype was examined by transfer of this mutation to a laboratory CMV strain (strain Towne). The recombinant virus was resistant to ganciclovir (8.4-fold increase in the 50% inhibitory concentration), was sensitive to foscarnet, and replicated normally in cell culture.

Identification of a functional Ca2+-sensing receptor in normal human gastric mucous epithelial cells.

The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]i were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]i in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.

Effects of paclitaxel on the growth of normal, polyposis, and cancerous human colonic epithelial cells.

BACKGROUND: The specific paclitaxel dose or time course in the treatment of colon carcinoma without the disruption of normal colonic cell proliferation is currently not known. The aim of this study was to determine the effects of paclitaxel on the growth of human colonic epithelial cells using cultures of normal, polyposis, and cancerous cells. METHODS: Normal, polyposis, and cancerous human colonic cells (Caco-2, T-84, and LoVo cell lines) were cultured, then treated with paclitaxel (10(-9)-10(-5) M) for 0-7 days.[AU: Please verify all dosages throughout.] Cell proliferation was assayed using either a Coulter-Counter or MTT-growth assay. Immunofluorescence and Western immunoblotting measured P-glycoprotein. RESULTS: Low paclitaxel doses (1 x 10(-9)-10(-8) M) were more effective than higher paclitaxel doses (>1 x 10(-8) M) in the growth inhibition of polyposis, Caco-2, and LoVo cancer (but not T-84) cell lines. Low paclitaxel doses had little effect on normal colonic cell growth over 7 days. Higher paclitaxel doses (>1 x 10(-8)-10(-5) M) produced a dose-dependent inhibitory effect on the growth of normal human colonic epithelial cells over 7 days but had no effect on the growth of polyposis, Caco-2, and LoVo cells over 3-7 days of treatment. Immunofluorescence and Western immunoblotting of cultures showed that 1 x 10(-6) M paclitaxel increased P-glycoprotein expression in Caco-2 and LoVo cells. There was no effect of paclitaxel on P-glycoprotein expression in T-84 cancer cells, which were found to have high endogenous basal levels of P-glycoprotein. P-glycoprotein expression in Caco-2 cells was found on plasma membranes and in perinuclear areas. CONCLUSIONS: Lower paclitaxel doses are more effective over time for the growth inhibition of polyposis and cancerous colonic cells, with minimal effects on the growth of normal colonic epithelial cells. Increased P-glycoprotein expression appears to be correlated with paclitaxel resistance in polyposis and cancerous colonic cells.

Establishment of human colonic epithelial cells in long-term culture.

Study of normal colonic function is important in understanding the cellular mechanisms of carcinogenesis and other diseases of the colon. However, colonic pathophysiological studies have been limited due to the lack of long-term cultures of normal human colonic epithelial cells. The purpose of the present study was to develop methods of isolating viable human colonic epithelial cells for the establishment of nontransformed colonic epithelial cell lines. Human colonic epithelial cells were isolated from surgically resected normal human colons. We found that the use of a short enzymatic digestion gave a consistently higher number (>90%) of viable human colonic epithelial cells. These isolated colonocytes were grown on plastic, collagen-coated filters, or feeder layers using different media formulations. Those colonocytes from the initial primary cultures that were most "epithelial" in appearance were cloned and passaged to establish long-term cultures of nontransformed human colonic epithelial cells. The epithelial nature and secretory function of these established cell lines were confirmed by morphological criteria (light microscopy,, phase contrast microscopy, and electron microscopy). We found that the long-term cultures remained immunopositive to anti-cytokeratin antibodies and immunonegative to anti-vimentin antibodies. Using a soft agar assay we found that the colonocytes did not form colonies, suggesting that the long-term culturing did not cause these cells to become transformed. Under serum-free conditions, we found that epidermal growth factor and transforming growth factor-alpha were equally potent in their mitogenic effects for these colonocytes. Some of the subcultured cells could be maintained for at least 8 months and still retain their epithelial characteristics. We believe that this methodology will serve as a valuable tool for the isolation and culturing of human colonic epithelial cells for studies of normal and malignant colonic disease processes.