PKCalpha/beta I inhibitor Go6976 induces dephosphorylation of constitutively hyperphosphorylated Rb and G1 arrest in T24 cells.

BACKGROUND: Rb functions as a key controller of the G(1)-S transition of the cell cycle, and its inactivation leads to a defective G(1) checkpoint. Bladder cancer frequently displays alterations in Rb such as constitutive hyperphosphorylation which results in inactive Rb and progression of cells to the S-phase. Several protein kinase C (PKC) inhibitors are currently undergoing clinical trials as anticancer drugs. MATERIALS AND METHODS: T24 urinary bladder carcinoma cells, known to express hyperphosphorylated Rb, were treated with PKCα/ßI inhibitor Go6976. The treated cells were subjected to cell cycle analysis, cell growth assay and Western blots for Rb and cdc2 phosphorylation. RESULTS: The treatment resulted in Rb dephosphorylation at Ser 795 and Ser 807/811, and cdc2 dephosphorylation at Tyr15. Subsequent G(0/1) arrest and reduced proliferation rates were observed. CONCLUSION: The results show that Go6976 can be used to restore constantly hyperphosphorylated and therefore constantly inactive Rb function in cancer cells.

Expression of cyclooxygenase-1 and -2 in urinary bladder carcinomas in vivo and in vitro and prostaglandin E2 synthesis in cultured bladder cancer cells.

Cyclooxygenases (Coxs) are the rate-limiting enzymes catalysing the formation of prostaglandins, which are involved in various of physiological processes. Increased Cox-2 expression has been observed in several malignancies, but the exact role of Cox-2 in carcinogenesis remains unsolved. We studied the expression of both Cox-1 and Cox-2 by immunohistochemistry in 29 transitional cell carcinomas of the urinary bladder. Diffuse cytoplasmic immunosignal for Cox-2 was detected in all cancer specimens. The expression was moderate in 55% and strong in 31% of the carcinomas. The normal urothelium in the samples stained also for Cox-2, but the intensity of the immunosignal was weak in most specimens. Cox-1 was expressed in the stroma of bladder wall, whereas in the tumour cells, Cox-1 immunosignal was either absent or weak. No correlation was detected between Cox-1 or Cox-2 expression and tumour differentiation or stage of invasion. We also evaluated the mRNA expression of Cox-1 and Cox-2 and synthesis of prostaglandin E2 (PGE2) in three bladder carcinoma cell lines (RT4, 5637, and T24). All cell lines expressed high levels of Cox-2 mRNA, whereas Cox-1 mRNA expression was detected only in T24 cells. There was great variation in the basal levels of PGE2 synthesis in these cell lines. Indomethacin inhibited the synthesis of PGE2 in all three cell lines, although the level of Cox-2 mRNA tended to increase by indomethacin. These results indicate that Cox-2 is widely expressed in human bladder carcinomas and that the role of Cox-2 inhibition in bladder cancer should be further studied.

Sucrose has no beneficial effects on wound healing in rats.

OBJECTIVE: To evaluate the effects of sucrose treatment on the formation of granulation tissue in a standard wound model. DESIGN: Animal study. SETTING: University hospital, Finland. ANIMALS: 32 male Sprague-Dawley rats divided into 4 groups. INTERVENTIONS: Implantation of viscose cellulose sponge subcutaneously, and daily injection of three concentrations of sucrose (0.01, 0.1 or 1 M) or vehicle for 7 days. MAIN OUTCOME MEASURES: The amount of granulation tissue measured by chemical analysis and histology. The amount and distribution of types I and III collagen assayed by immunofluorescence. RESULTS: None of the three concentrations altered the amounts of DNA, RNA, hydroxyproline, nitrogen, hexosamines, and uronic acids in granulation tissue. Neither improvement nor deterioration was seen in the growth of granulation tissue in histological specimens. The amount and distribution of types I and III collagen was similar in controls and sucrose-treated rats. Type III collagen was most abundant near newly-formed vessels. Neither sucrose nor fructose was found in wound fluid while the concentration of glucose was significantly lower in all test groups than in controls. CONCLUSIONS: Sucrose solution had neither beneficial nor deleterious effects on the amount of developing granulation tissue in an experimental wound model. The amount and distribution of types I and III collagens were also not altered by sucrose treatment.

Expression of collagenase-3 (matrix metalloproteinase-13) in transitional-cell carcinoma of the urinary bladder.

Expression of collagenase-3 [matrix metalloproteinase-13 (MMP-13)] has been previously demonstrated in squamous-cell carcinomas of both the head and neck and the vulva, cutaneous basal-cell carcinomas, chondrosarcomas and melanomas. Using in situ hybridization, MMP-13 mRNA expression was detected in 13 of 23 (52%) urinary bladder transitional-cell carcinomas (TCCs). Expression was restricted to cells in the invading edges of tumors. No expression of MMP-13 mRNA could be detected in normal urothelium. As detected by immunohistochemistry, MMP-13 protein showed an expression pattern similar to that of MMP-13 mRNA. Expression of MMP-13 mRNA and protein was also detected in 2 bladder carcinoma cell lines (RT4 and T24). In these cell lines, TNF-alpha potently induced MMP-13 mRNA expression. Retinoids and a selective p38 inhibitor, SB203580, potently inhibited MMP-13 mRNA expression. Our results demonstrate MMP-13 expression in human urinary bladder carcinoma cells in vivo and in vitro and suggest that MMP-13 may serve as a marker for transformation and invasion in urinary bladder TCCs.

Wound healing in denervated rat groin skin flap.

The purpose of the present study was to investigate the effect of denervation on dermal wound healing in rat groin skin flaps for 1-10 weeks. The structural differences between wounds in normal and in denervated skin were investigated histologically using Herovici's staining. Pro alpha1(I) collagen mRNA levels were studied using Northern hybridization. Denervation and reinnervation of the skin flaps was demonstrated with quantitative noradrenaline determination and immunohistochemically using neurofilament and S-100 antibodies. Denervation of the skin did not seem to have any apparent effects on wound healing as assessed by light microscopy. There were no significant differences in pro alpha1(I) collagen mRNA levels either. The thin muscle layer underlying the skin was the only element that clearly responded to the denervation.

Urinary bladder transitional cell carcinogenesis is associated with down-regulation of NF1 tumor suppressor gene in vivo and in vitro.

The NF1 gene product (neurofibromin) is known to act as a tumor suppressor protein by inactivating ras. The best documented factors involved in urinary bladder transitional cell carcinoma (TCC) are ras proto-oncogene activation and p53 suppressor gene mutations. This is the first study reporting alterations in NF1 gene expression in TCC. We examined NF1 gene expression in a total of 29 surgical urinary bladder TCC specimens representing grades 1 to 3 and in three cell lines, RT4, 5637, and T24 (representing grades 1 to 3, respectively). Decreased NF1 gene expression was observed in 23 of 29 (83%) TCC specimens as estimated by immunohistochemistry, the decrease being more pronounced in high-grade tumors. NF1 mRNA levels were markedly lower in TCC tissue compared with adjacent non-neoplastic urothelium, as studied by in situ hybridization for grade 3 TCC. Immunohistochemistry and Western blotting demonstrated that TCC cell lines expressed NF1 protein at different levels, expression being almost undetectable in T24 (grade 3) cells. Northern blotting for cell lines demonstrated reduced NF1 mRNA levels in grade 3 TCC cells. Reverse transcription polymerase chain reaction for cell lines and selected grade 2 and grade 3 tissue samples demonstrated NF1 type II mRNA isoform predominance in all samples studied. Our results show that both NF1 mRNA and protein levels are decreased in high-grade TCC, suggesting that alterations of NF1 gene expression may be involved in bladder TCC carcinogenesis.

Upregulation of tumor suppressor protein neurofibromin in normal human wound healing and in vitro evidence for platelet derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) elicited increase in neurofibromin mRNA steady-state levels in dermal fibroblasts.

We first studied expression of neurofibromin by immunohistochemistry in scars obtained from operations involving areas of healing wounds. The results demonstrated increased immunoreactivity for neurofibromin in the fibroblastic cell population of the lesions when compared with fibroblasts of apparently healthy perilesional skin, or those of intact control skin. Furthermore, dermal fibroblasts of 19 and 34 wk-old fetuses displayed a clearly detectable immunosignal for neurofibromin. In vitro studies were designed to investigate the potential effects of selected growth factors--known to be operative in wound healing--on neurofibromin mRNA steady-state levels in cultured fibroblasts. Northern transfer analyses revealed that different isoforms of platelet derived growth factor (PDGF) exerted selective effects on the neurofibromin mRNA levels: PDGF isoform AB elevated neurofibromin mRNA levels in a concentration-dependent manner when concentrations of 0.1, 1, 10, and 30 ng per ml were used. The maximal upregulatory effect of PDGF BB was reached at a concentration of 1 ng per ml. In contrast, PDGF AA did not alter the steady-state levels of neurofibromin mRNA. As estimated by RNase protection assay, transforming growth factor-beta1 (TGF-beta1) upregulated neurofibromin gene expression when concentrations of 0.5 and 5 ng per ml were used. Reverse transcription followed by polymerase chain reaction did not detect apparent alterations in the ratio of type I/type II neurofibromin isoforms in PDGF- or TGF-beta1-treated cultures. Taken together, our results suggest that expression of tumor suppressor protein neurofibromin is upregulated in response to skin injury, and that this upregulation can be mediated through PDGF and TGF-beta.

Developmental regulation of NF1 tumor suppressor gene in human peripheral nerve.

Mutations of the NF1 tumor suppressor gene cause type 1 neurofibromatosis, characterized by multiple tumors of the peripheral nerves, as well as other tumor types. The NF1 protein, neurofibromin, is intricately linked to the cell growth regulatory signalling pathways, e.g. by possessing RAS-GTPase activity. The regulation and role of neurofibromin are not known in normal human development. We addressed this issue by studying the regulation of neurofibromin in normal human peripheral nerves, from early fetal development to adulthood. The barely detectable neurofibromin immunosignal in peripheral nerves during the first trimester of gestation contrasted dramatically to its increase in Schwann cells, perineurial cells, and axons during the second and third trimesters. Interestingly, the type I and II isoforms of neurofibromin, differing in their RAS oncoprotein inactivation capacity, displayed clearly different expression profiles throughout these periods. This suggests distinct cellular functions for these neurofibromin isoforms. The results also revealed distinct species-specific differences in neurofibromin expression, potentially bearing relevance to the lack of human neurofibromatosis-like disorders in other species.