MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma.

Mammalian sirtuin 1 (SIRT1) has connected to an ever widening circle of activities that encompass cellular stress resistance, energy metabolism and tumorigenesis. However, underlying mechanisms leading to oncogenic SIRT1 overexpression are less understood. In this study, we identified SIRT1 regulatory microRNA (miRNA) and its function in hepatocellular carcinoma (HCC). Aberrant SIRT1 overexpression was demonstrated in a subset of human HCCs. SIRT1 knockdown suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins. This led to hypophosphorylation of pRb, which inactivated E2F/DP1 target gene transcription, and thereby caused significant increase of HCC cells to remain in the G1/S phase. A comprehensive miRNA profiling analysis indentified five putative endogenous miRNAs that are significantly downregulated in HCC. Ectopic expression of miRNA mimics evidenced miR-29c to suppress SIRT1 in HCC cells. Notably, ectopic miR-29c expression repressed cancer cell growth and proliferation, and it recapitulated SIRT1 knockdown effects in HCC cells. In addition, miR-29c expression was downregulated in a large cohort of HCC patients, and low expression of miR-29c was significantly associated with poor prognosis of HCC patients. Taken together, we demonstrated that miR-29c suppresses oncogenic SIRT1 by way of binding to 3'-untranslated region of SIRT1 mRNA causing translational inhibition in liver cancer cells. The loss or suppression of miR-29c may cause aberrant SIRT1 overexpression and promotes liver tumorigenesis. Overall, we suggest that miR-29c functions as a tumor suppressor by regulating abnormal SIRT1 activity in liver.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line.

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Opportunistic hepatic infections in AIDS patients with fever of unknown origin.

The clinical features and histopathologic manifestations of hepatic opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS) in Taiwan remain unexplored. We report 28 AIDS patients (25 men, 3 women; mean age, 34 years) with fever of unknown origin who underwent 31 liver biopsies from December 1995 to May 1997. In most cases, the biochemical tests showed moderate to markedly elevated alkaline phosphatase concentrations, but normal or mildly elevated aminotransferase concentrations. The most common histopathologic finding was macrosteatosis, which was noted in 15 of the 28 patients. Another important histopathologic finding indicating the etiology of hepatic opportunistic infection was granuloma, which was found in 11 patients. Histochemical stain and culture of liver specimens yielded Mycobacterium avium complex (MAC) in eight patients, Mycobacterium tuberculosis in two patients, Histoplasma capsulatum in one patient, and cytomegalovirus in one patient with concomitant MAC infection. Therefore, a definitive diagnosis in AIDS patients with fever of unknown origin was made in 11 of the 28 cases with the assistance of liver biopsy. During follow-up, late extrahepatic involvement by the same infectious agents was found in six patients. Thus, hepatic manifestations could be a harbinger of disseminated opportunistic infections in AIDS patients.

PIP: AIDS patients are open to many opportunistic infections which often present as fever. The cases of 25 men and 3 women with AIDS of mean age 34 years, with fever of unknown origin who underwent 31 liver biopsies in Taiwan from December 1995 to May 1997, are presented. The biochemical tests most often showed moderate to markedly elevated alkaline phosphatase concentrations, but normal or mildly elevated aminotransferase concentrations. The most common histopathologic finding was macrosteatosis, noted in 15 of the 28 patients, while granuloma was found in 11 patients. The histochemical stain and culture of liver specimens yielded Mycobacterium avium complex (MAC) in 8 patients, Mycobacterium tuberculosis in 2 patients, Histoplasma capsulatum in 1 patient, and cytomegalovirus in 1 patient with concomitant MAC infection. A definitive diagnosis in AIDS patients with fever of unknown origin was therefore made in 11 of the 28 cases with the assistance of liver biopsy. During follow-up, late extrahepatic involvement by the same infectious agents was found in 6 patients. Hepatic manifestations could therefore be a harbinger of disseminated opportunistic infections in AIDS patients.

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression.

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.

Color stability of composite resins.

The color stability of seven visible light-cured and three chemically-cured composite resins was investigated while being subjected to UV light irradiation and storage in an aqueous environment at elevated temperatures. Color shift was evaluated visually and by colorimetric measurements. Significant correlation was found between visual scoring and colorimetric readings. When subjected to UV light, a wide deviation in color change existed from brand to brand in photocured composite resins. The color shift of chemically-cured composite resins was less than but fell within the range of photocured composite resins. When stored in water at elevated temperatures, photocured resins exhibited better color stability than the chemically-cured composite resins.