Significance of target cell infection and natural killer cells in the anti-tumor effects of bacillus Calmette-Guerin in murine bladder cancer.

Although intravesical instillation of bacillus Calmette-Guerin (BCG) is a clinically well-recognized therapy for bladder carcinoma in situ and recurrence prophylaxis, these mechanisms have not been fully understood. We studied the effects of BCG infection (Connaught strain) on target cancer cells and host immune systems in murine bladder cancer. The bladder cancer cell line, MB49, was used in C57/BL6 mice in vivo and in vitro. In vitro cytotoxicities against the cancer cell line were measured by 24-h 51Cr release assay. For effector cells, spleen mononuclear cells were obtained from mice injected intraperitoneally with BCG or BCG-infected irradiated MB49 cells. Although BCG infection of cancer cells did not affect the proliferation speed in vitro, the mice injected subcutaneously with BCG-infected MB49 cells survived significantly longer than those given untreated cancer cells. The mice surviving without tumor growth after injection of BCG-infected cancer cells could not reject a second injection of intact MB49 cells. In vitro cytotoxicity was enhanced by BCG infection of target cancer cells, but not by immunizing the mice with BCG from which effector cells were obtained. Moreover, cytotoxicity disappeared by depleting natural killer (NK) cells from effector cells. Although in vitro cytotoxicity was increased by immunizing the mice with BCG-infected irradiated MB49 cells, survival did not improve in these mice. These results suggest that a major part of BCG's anti-tumor effects can be attributed to the elimination of BCG-infected cancer cells by NK cells.

Norepinephrine activates P44 and P42 MAPK in human prostate stromal and smooth muscle cells but not in epithelial cells.

BACKGROUND: In vascular smooth muscle cells, alpha1-adrenergic stimulation increases DNA synthesis and cell proliferation via activation of p44/42 (ERK1/2) MAPK. We examined whether norepinephrine (NE) activates MAPK and stimulates the proliferation of prostatic epithelial and non-epithelial cells. METHODS: Human prostatic epithelial cells, stromal cells, and smooth muscle cells were purchased from BioWhittaker (Walkersville, MD). After reaching a semi-confluent condition, the cells were cultured in RPMI-1640 without serum for 1 day. At 10 min after adding NE (10(-6) or 10(-7) M) to the medium, the cells were collected. Cell lysate was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot using anti-phospho-p44/42 and anti-p44/42 antibodies. The activation of p44/42 was estimated by the ratio of phospho-p44/42 to total p44/42. Cell proliferation was evaluated by (3)H-thymidine uptake assay. After reaching a semi-confluent condition, the cells were cultured in RPMI-1640 containing 0.5% FCS with or without NE (10(-6) or 10(-7) M) for 16 hr followed by a (3)H-thymidine uptake period (24 hr). RESULTS: P44/42 MAPK was significantly activated by NE in non-epithelial cells (stromal cells and smooth muscle cells) while not in epithelial cells. The uptake of (3)H-thymidine was significantly increased by NE in both non-epithelial cells, which was inhibited by alpha1-adrenoceptor antagonists. CONCLUSIONS: These results suggest that NE may stimulate the proliferation of non-epithelial prostatic cells, which may be involved in the pathogenesis of BPH.

Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney.

BACKGROUND: The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method. METHODS: Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened. RESULTS: The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. CONCLUSIONS: Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.