Holliday junction­recognition protein modulates apoptosis, cell cycle arrest and reactive oxygen species stress in human renal cell carcinoma.

Holliday junction recognition protein (HJURP) is involved in the regulation of mortality in various cell types, including renal cell carcinoma (RCC) cells. The specific mechanisms by which HJURP regulates RCC cell apoptosis and the cell cycle have not been previously investigated, to the best of our knowledge. In the present study, the expression of HJURP in RCC tissues and adjacent paracancerous renal tissue, as well as in RCC cell lines, was analyzed using reverse transcription­quantitative PCR and western blot analysis. The A498 RCC cells were transfected with an HJURP overexpression vector, which resulted in reduced proliferation, as demonstrated using immunofluorescence staining, a Cell Counting Kit­8 assay and a colony formation assay. Flow cytometry and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling assays were used to determine the effect of HJURP on the cell cycle and apoptosis of RCC cells. Proteins associated with the reactive oxygen species (ROS) status were analyzed using western blot analysis. The expression of HJURP was lower in RCC tissues and cells compared with that in the adjacent paracancerous renal tissues and control cells. Furthermore, overexpression of HJURP resulted in a decrease in cell viability and proliferation in vitro. Overexpression of HJURP resulted in cell cycle arrest at the G0/G1 phase, cell apoptosis and an increase in ROS stress. In addition, the phosphorylated/total sirtuin 1 (SIRT1) protein ratio was decreased, whereas the expression of peroxisome proliferator­activated receptor (PPAR)γ was increased in the HJURP­overexpressing RCC cells. In clinical practice, decreased HJURP expression may be associated with poor prognosis in patients with RCC. These results suggest that HJURP may regulate cell apoptosis and proliferation in RCC cells and this may be mediated by PPARγ/SIRT1. Thus, HJURP may be used as a predictor of prognosis in patients with RCC.

Quantitative risk stratification and individual comprehensive therapy for invasive bladder cancers in China.

BACKGROUND: To evaluate the risk factors for invasive bladder cancer and to develop a predictive model for the improvement of individual comprehensive therapy for invasive bladder cancers. MATERIALS AND METHODS: The records of 356 patients with invasive bladder cancer, operated on at three Chinese medical institutes, were reviewed. The Cox proportional hazards regression model was used to assess the clinical and pathological variables affecting disease-free survival (DFS). The regression coefficients determined by Cox regression analysis were used to construct a predictive index (PI). PI was used to categorize the patients into different risk groups. Kaplan-Meier survival curves followed with log-rank test were plotted to compare the difference. RESULTS: Tumor configuration (RR = 1.60, P = 0.01), multiplicity (RR = 1.41, P = 0.04), histological subtype (RR = 2.13, P < 0.01), tumor stage (RR = 2.50, P < 0.01), tumor grade (RR = 2.35, P < 0.01), node status (RR = 2.48, P < 0.01), and neoadjuvant chemotherapy (RR = 0.46, P = 0.02), had independent prognostic significance for DFS. PI = 0.47 x (configuration) + 0.34 x (multiplicity) + 0.76 x (tumor histological subtype) + 0.92 x (stage) + 0.86 x (grade) + 0.91 x (node status) - 0.79 x (neoadjuvant chemotherapy). The range of PI was -0.32 to 6.52, which was equally divided into three risk groups with significant differences on Kaplan-Meier curves and a log-rank test (P < 0.01). Meanwhile, the patient's probability of survival could be calculated by PI. CONCLUSIONS: Seven factors (tumor configuration, multiplicity, histological subtype, tumor stage, tumor grade, node status, neoadjuvant chemotherapy) affect the prognosis after radical cystectomy (RC) for invasive bladder cancer. PI can be used to optimize the individual comprehensive therapy. Given fewer perioperative complications, fast recovery from surgery and relatively satisfactory quality of life, ureterocutaneostomy, and ileal conduit are suitable for the patients with short expected life spans.

[Relation between activation of NF-kappa B and chemotherapy induced apoptosis of leukemic cells].

OBJECTIVE: To analyze the relation between activation of NF-kappa B and chemotherapy induced apoptosis of leukemic cells and the effect of vincristine (VCR) on them. METHODS: Electrophoretic mobility shift assay (EMSA) was used to detect the activation of NF-kappa B and tunel DNA electrophoresis was adopted to observe the apoptosis induced by cytosine arabinoside (Ara-C) and etopside (Vp-16) in P388 leukemic cells. RESULTS: The activation of NF-kappa B induced by Ara-C and Vp-16 was obviously correlated to apoptosis in P388 cells. VCR (0.1 micromol/L) could suppress activation of NF-kappa B by 52% and 63% and significantly increase the apoptosis by 89% and 123% as induced by Ara-C (100 micromol/L) and Vp-16 (100 micromol/L). The activity of NF-kappa B could be found in P388 cells before being exposed to chemotherapeutic agent. CONCLUSION: Chemotherapeutic agents can induce apoptosis and activation of NF-kappa B of P388 cells. The mechanism of VCR potentiating chemotherapeutics induction of leukemia cell apoptosis may be related to its suppression of the NF-kappa B activity in the P388 cells.

[Inhibition of activation of nuclear factor-kappaB enhanced apoptosis of leukemic cells induced by homoharringtonine].

OBJECTIVE: To explore the effects of dexamethasone (DXM) and vincristine (VCR) on apoptosis of K562-n cells and activation of nuclear factor-kappaB-gene binding (NF-kappaB) in K562-n cells induced by homoharringtonine. METHODS: K562-n cells were cultured in RPMI-1640 medium. Homoharringtonine of various concentrations was added into the cultures. Twelve hours later, the apoptosis induced by homoharringtonine in K562-n cells was analysed by TdT-mediated X-dUTP nick and end labeling (TUNEL) and DNA electrophoresis. Another sample of K562-n cells was culture together with DXM (1 micro mol/L) or VCR (0.1 micro mol/L) for 2 hours, then homoharringtonine was added into the cultures. Twelve hours later, the apoptosis in K562-n cells was analysed by TUNEL and DNA electrophoresis. Still another sample of K562-n cells was cultured for 3 hours with homoharringtonine, then electrophoretic mobility shift assay (EMSA) was conducted to determine the DNA-binding activation of NF-kappaB. A fourth sample of K562-n cells was cultured together with DXM (1 micro mol/L) or VCR (0.1 micro mol/L) for 2 hours, then homoharringtonine was added into the cultures. Three hours later, EMSA was conducted. RESULTS: The apoptosis rates of K562-n cells induced by homoharringtonine of various concentrations were (30.00 +/- 3.34)%, (47.13 +/- 3.18)% and (68.63 +/- 8.14)%, respectively. The apoptosis rates of K562-n cells induced by homoharringtonine being pre-processed with DXM or VCR were (55.75 +/- 3.88)% and (64.38 +/- 4.60)%, respectively, being 85.8% and 114.6% higher than that induced by homoharringtonine alone (30.00 +/- 3.34)% (all P < 0.05). Activation of NF-kappaB in K562-n cells was induced significantly by homoharringtonine. Activation of NF-kappaB in K562-n cells induced by homoharringtonine could be suppressed by being pre-processed with 1.0 micro mol/L DXM or 0.1 micro mol/L VCR. The rate of suppression was 32.0% and 39.4% respectively. CONCLUSION: Homoharringtonine induces apoptosis of K562-n cells and induces NF-kappaB activation in K562-n cells. The mechanism of increased apoptosis of K562-n cells with DXM or VCR may be related to suppression of activation of NF-kappaB of K562-n cells.

Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression.

OBJECTIVE: To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. METHODS: The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. RESULTS: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation. CONCLUSIONS: These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.