[Vincristine inhibits the proliferation of ovarian cancer cells by regulating the demethylation of RASSF2A].

Objective: To investigate the effect of vincristine on the proliferation of ovarian cancer cells by regulating RASSF2A demethylation. Methods: SKOV3 cells were infected with control (LV-NC) and RASSF2A lentivirus (LV-RASSF2A) and treated with or without vincristine. Cell counting kit-8 (CCK-8) was used to detect the activity of ovarian cancer cells (SKOV3) treated with different doses of vincristine. Colony formation assay was used to detect the proliferation of SKOV3 cells. Flow cytometry was used to detect the apoptosis of SKOV3 cells. Real time polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of RASSF2A in IOSE-29 and SKOV3 cells. Western blot was used to examine the protein expression of RASSF2A in IOSE-29 and SKOV3 cells. Methylation-specific PCR was used to detect methylation and demethylation levels of RASSF2A gene in IOSE-29 and SKOV3 cells. Results: The cell viabilities of SKOV3 cell treated with 6.25 nmol/L, 12.5 nmol/L, 25 nmol/L, 50 nmol/L and 100 nmol/L vincristine were (87.19±4.49)%, (73.67±8.62)%, (66.35±6.04)%, (50.32±6.00)% and (34.92±6.11)%, respectively, lower than (100.46±4.69)% of control group (P<0.05). The half maximal inhibitory concentration of vincristine at 48 hours was 50.02 nmol/L. The proliferation abilities of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (41.70±2.21)%, (32.15±1.80)% and (23.00±2.01)%, respectively, significantly lower than (100.78±5.66)% in the control group (all P<0.05). The apoptotic rates of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (3.65±0.27)%, (5.21±0.76)% and (10.46±1.00)%, respectively, significantly higher than (2.12±0.23)% in the control group (all P<0.05). Compared with the IOSE-29 group (1.00±0.07 and 0.68±0.04), the mRNA expression (0.32±0.04) and protein expression (0.24±0.02) of RASSF2A were down-regulated in SKOV3 cells (P<0.05). Compared with the LV-NC group [(101.60±4.39)%, (100.73±3.29)%, (4.06±0.30)%], over-expression of RASSF2A down-requlated cell viability (68.92±3.94)%, inhibited proliferation (16.38±2.16)%, and promoted apoptosis (8.65±0.56)%, (P<0.05). Conclusion: Vincristine can increase RASSF2A expression and inhibit ovarian cancer cell proliferation by promoting the demethylation of RASSF2A promoter.

[Notch signaling pathway participates in the differentiation of hepatic progenitor cells into bile duct epithelial cells and progression of hepatic fibrosis in cholestatic liver fibrosis rat].

Objective: To investigate differentiation direction of hepatic progenitor cells (HPCs) in cholestatic liver fibrosis (CLF), and the role of Notch signaling pathway in the differentiation of HPCs. Methods: A CLF rat model was established by bile duct ligation (BDL) followed by monitoring changes of Notch signal pathway and the cellular origin of proliferating cholangiocytes. After intraperitoneal injection of DAPT (a Notch signaling inhibitor) after bile duct ligation, the progress of liver fibrosis and the proliferation of cholangiocytes after inhibition of the Notch pathway were analyzed. Results: Data showed that bile duct proliferation gradually increased along with inflammatory cell infiltration and proliferating bile duct cells surrounded by abundant collagen in the BDL group. Immunostaining confirmed markedly increased expression of CK19, OV6, Sox9 and EpCAM. In addition, RT-PCR results showed that Notch signaling pathway was activated significantly. Once the Notch signaling pathway was inhibited by DAPT, bile duct proliferation markedly suppressed along with significantly decreased the mRNA expression of CK19, OV6, Sox9 and EpCAM, compared with BDL group [(10.2±0.7) vs. (22.3±0.8), (7.6±1.5) vs. (18.1±3.7), (1.4±0.4) vs. (4.1±1.1), (1.3±0.3) vs. (5.0±1.4), respectively, P<0.01]. Moreover, liver fibrosis was also reduced significantly. Conclusion: Notch signaling activation is required for HPCs differentiation into cholangiocytes in CLF and inhibition of the Notch signaling pathway may offer a therapeutic option for treating CLF.

Hypermethylation of hepatic Gck promoter in ageing rats contributes to diabetogenic potential.

AIMS/HYPOTHESIS: Hepatic glucokinase (GCK) is a key enzyme in glucose utilisation. Downregulation of its activity is associated with insulin resistance and type 2 diabetes mellitus. However, it is unknown whether hepatic Gck expression is influenced by age and is involved in ageing-mediated diabetes, and whether the degree of methylation of the hepatic Gck promoter is correlated with the transcription of Gck. To address the question, we evaluated hepatic Gck transcription and promoter methylation in young (14 weeks), adult (40 weeks) and aged (80 weeks) rats. METHODS: Hepatic glycogen, Gck expression and the kinase activity of GCK were measured in three age groups. The CpG methylation status was determined by both bisulphite direct sequencing and clone sequencing of the PCR amplificates of Gck promoter. The causal relationship between Gck methylation and mRNA expression was confirmed by treating rat primary hepatocytes with 5-aza-2'-deoxycytidine (5-Aza-CdR). RESULTS: We have shown an age-associated decline in hepatic glycogen, Gck expression levels and the kinase activity of hepatic GCK. The eleven CpG sites studied displayed age-related progressive methylation changes in hepatic Gck promoter, which were confirmed by two methods: direct and clone sequencing. After 5-Aza-CdR treatment of rat primary hepatocytes, there was a fourfold increase in Gck expression. CONCLUSIONS/INTERPRETATION: Our results demonstrate that an age-related increase in methylation is negatively associated with hepatic Gck expression, suggesting that DNA methylation could be involved in increasing age-dependent susceptibility to hepatic insulin resistance and diabetes. Thus, the epigenetic modification of the hepatic Gck promoter may represent an important marker for diabetogenic potential during the ageing process.

Integrin-mediated interactions between primary/T-sv40 immortalized human glomerular epithelial cells and type IV collagen.

The use of human glomerular epithelial cells (HGEC) in research has been severely restricted by several obstacles, which have been circumvented by the generation of T-SV40 immortalized human visceral glomerular epithelial cells (Delarue et al, 1991). In this work, we compared the primary and immortalized HGEC for expression of integrin and some nonintegrin surface receptors. We also studied the adhesion of both types of HGEC to glomerular basement membrane (GBM), type IV collagen (tIV), and its major noncollagenous NC1 domain. The integrins mediating adhesion of HGEC to tIV were also examined. Expression of integrin and some nonintegrin cell surface receptors was analyzed by flow cytometry. Adhesion to GBM, tIV, and its major noncollagenous NC1 domain was studied by direct solid phase cell adhesion assays. Identification of integrins mediating adhesion of HGEC to tIV was achieved by inhibition of cell adhesion using monoclonal antibodies to integrin subunits. The primary and immortalized HGEC share phenotypic characteristics, and alpha3beta1 appeared to be the major integrin present on both HGEC types. The kinetics of binding to GBM, tIV, and its noncollagenous NCI domain were similar in both the primary and immortalized HGEC, although the latter displayed a somewhat weaker binding. Both the primary and immortalized HGEC displayed significantly better adhesion to NC1-alpha3 compared with NC1-alpha1, alpha3beta1 appears to be the major integrin mediating the adhesion of HGEC to tIV. Our studies suggest that alpha3beta1 is the major integrin present on HGEC. This has been confirmed by flow cytometric analysis. In addition, we demonstrated a functional role for this integrin in mediating attachment of HGEC to tIV. Our data also demonstrate a preference in binding of HGEC to alpha3 chains of NC1 compared with alpha1 chains of NC1. These findings were seen in both the primary and immortalized HGEC. The T-SV40 immortalized HGEC can therefore serve as a very useful tool to study glomerular visceral cell biology.

Immunochemical studies of the Alport antigen.

The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.

Nucleus-dependent regulation of tyrosine aminotransferase degradation in hepatoma tissue culture cells.

Upon removal of the nucleus from rat hepatoma tissue culture cells, levels of the enzyme tyrosine aminotransferase no longer change in response to withdrawal of glucocorticoids. The rate of tyrosine aminotransferase degradation is drastically reduced in rat hepatoma tissue culture cytoplasts leading to stabilization of pre-existing levels of tyrosine aminotransferase. Moreover, the rate of synthesis of the enzyme in cytoplasts is very low near that observed in uninduced whole cells. These effects of enucleation occur very rapidly and appear to be specific for tyrosine aminotransferase and a small number of other unstable hepatoma proteins. A nuclear effect is thus directly involved in the control of tyrosine aminotransferase degradation and synthesis.