Vesa Aaltonen

Protein Kinase C α/β Inhibitor Go6976 Promotes Formation of Cell Junctions and Inhibits Invasion of Urinary Bladder Carcinoma Cells

Changes in activation balance of different protein kinase C (PKC) isoenzymes have been linked to cancer development. The current study investigated the effect of different PKC inhibitors on cellular contacts in cultured high-grade urinary bladder carcinoma cells (5637 and T24). Exposure of the cells to isoenzyme-specific PKC inhibitors yielded variable results: Go6976, an inhibitor of PKCα and PKCβ isoenzymes, induced rapid clustering of cultured carcinoma cells and formation of an increased number of desmosomes and adherens junctions. Safingol, a PKCα inhibitor, had similar but less pronounced effects. In contrast, a PKCδ inhibitor, rottlerin, had an opposite effect on cell clustering and caused dissociation of cell junctions. A broad-spectrum PKC inhibitor bisindolylmaleimide I did not have any apparent effect on the morphology of the cultures or on the number of cell junctions. Additional studies with Go6976 demonstrated that inhibition of PKCα and β isoenzymes induced translocation of β1-integrin from the cell-matrix junctions and that β4-integrin was translocated to face the culture substratum. Go6976 was also highly effective in inhibiting migration of carcinoma cells and inhibited invasion through artificial basement membrane. Our results on urinary bladder carcinoma cells emphasize that Go6976 is a potential anticancer drug due to its effects on cell-cell and cell-matrix junctions, migration, and invasion. Furthermore, the results may be explained by changes in PKC activation balance promoted by inhibition of PKCα/β.

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‐α 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. Int. J. Cancer 88:417–423, 2000.

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

Summary 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 Cox1 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 Cox2 expression and tumour differentiation or stage of invasion. We also evaluated the mRNA expression of Cox1 and Cox‐2 and synthesis of prostaglandin E 2 (PGE 2 ) 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 PGE 2 synthesis in these cell lines. Indomethacin inhibited the synthesis of PGE 2 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.

Heterogeneity of Cellular Proliferation within Transitional Cell Carcinoma: Correlation of Protein Kinase C Alpha/betal Expression and Activity

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-α and -βI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-α in 13 out of 18 samples and -βI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-α and/or -βI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-α was located in the cytoplasm, whereas PKC-βI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-α and -βI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

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.

Impaired gap junction formation and intercellular calcium signaling in urinary bladder cancer cells can be improved by Gö6976.

Purpose: Calcium wave propagation and connexin 26, 32 and 43 expression were studied in normal and malignant urothelial cells. Materials and Methods: Human urothelial cell cultures were established from tissue biopsies obtained from three healthy control persons and compared to human transitional cell carcinoma (TCC) cell line 5637. Fluo-3 was used to study intercellular calcium signaling in urothelial cells. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in intercellular calcium signaling. In addition, Gö6976 was used to determine the effects of PKC α and βI inhibition on intercellular calcium signaling. Results: In normal urothelial cells, the primary pathway for intercellular calcium mediated cell signaling was gap junctional intercellular communication (GJIC), but the paracrine ATP-mediated signaling was also operative. In 5637 TCC cells, GJIC and ATP-mediated signaling routes were altered when compared to normal urothelial cells. More specifically, inhibition of GJIC resulted in a complete block of intercellular calcium signaling, while inhibition of ATP-mediated signaling decreased signal transduction in 5637 TCC cells. The results of the present study also demonstrated that connexin 26 was the most abundant gap junction plaque protein in cultured normal human urothelial cells and that it did not form gap junction plaques in 5637 TCC cell culture. Treatment with Gö6976 induced gap junction plaque formation by connexin 26 in 5637 TCC cells. In addition, the exposure to Gö6976 enhanced intercellular calcium mediated signaling in 5637 TCC cells, but not in normal cells. Conclusions: The results of the present study suggest that gap junctions play a major role in intercellular calcium signaling in urothelial cells. In addition, intercellular calcium signaling is altered in urinary bladder carcinoma cells, and it can be improved by PKC α and βI inhibition. (Supplementary materials are available for this article. Go to the publishers online edition of Cell Communication and Adhesion for the following free supplemental resources; Movie files of normal Gö6976−, normal Gö6976+, TCC Gö6976−, TCC Gö6976+ and image of Supplementary ).

The Finnish national guideline for diagnosis, treatment and follow‐up of patients with wet age related macular degeneration

Age‐related macular degeneration (AMD) is the main cause of visual impairment in developed countries. Several improvements in the visualization of posterior segment of the eye together with the introduction of intravitreal anti‐VEGF treatment have revolutionized the prognosis of the wet form of AMD (wAMD). Increasing incidence of wAMD together with the limited resources of society and of the healthcare system poses challenges for the provision and development of care. In context of these current aspects, we aimed to set evidence‐based medical guidelines for diagnosis, treatment and follow‐up of patients with wAMD.

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.

Hypoxic conditions stimulate the release of B-type natriuretic peptide from human retinal pigment epithelium cell culture

A‐type peptide, a natriuretic peptide belonging to the natriuretic peptide family, has been shown to be increased in the vitreous of patients suffering from diabetic retinopathy and that human retina has a well‐developed natriuretic peptide system. The stimulus to which the synthesis of natriuretic peptides responded in these patients has, however, remained unknown. As the natriuretic peptides have recently been shown to respond to hypoxic conditions, the genes of both A‐type and B‐type have a hypoxia‐response element (HRE) in their promoter sequence, we therefore hypothesized that hypoxia in the human retinal pigment epithelium will increase the secretion of NT‐proBNP, the most common natriuretic peptide monitored in clinical medicine.

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.