Study of apoptosis gene expression in U937 cells induced by adhesion culture with mesenchymal stem cell / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare apoptosis gene expression profiling of U937 cells in suspension culture with that cultivated with mesenchymal stem cells (MSCs), and find out the relationship between drug resistance of leukemia cells and hemopoietic microenvironment.</p><p><b>METHODS</b>U937 cells were cultivated in adhesion culture with MSCs and in suspension culture for 48 hours. Cell cycle was determined by flow cytometry and gene expression profiling by cDNA microarray.</p><p><b>RESULTS</b>Compared with that in suspension, G(0)/G(1) fraction of U937 cells increased in adhesion culture (45.3 +/- 3.1)% vs (32.6 +/- 2.1)%, respectively (P < 0.05), whereas G(2)/M fraction and apoptosis rate were decreased. After 48 h twenty-eight differential expression genes were screened out in 487 apoptosis-related genes, among which 27 were up-regulated and were mainly apoptosis-suppressor genes, apoptosis-promoter genes, cell cycle positive control genes and cell cycle negative control genes. But Bcl-XL was up-regulated most obviously. The only one gene down-regulated was an apoptosis promoter gene.</p><p><b>CONCLUSION</b>Adhesion culture with MSCs can lead to growth suppression and decrease natural apoptosis of U937 cells. The mechanism was multiple gene effects, but Bcl-XL may be of the most importance.</p>

Study on the bone marrow mesenchymal stem cells induced drug resistance in the U937 cells and its mechanism / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The hematopoietic microenvironment (HM) plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of bone marrow derived mesenchymal stem cells (MSCs) in U937 cell line, to find out the relations between leukemia drug resistance and the MSCs.</p><p><b>METHODS</b>U937 cells were cultured in suspension or grew adherently with MSCs. The cell growth curve was drawn and the cell cycle was measured by flow cytometry. Apoptosis and sensitivity of U937 to daunoblastina (DNR) were quantified by DNA ladder detection and trypan blue exclusion assays, respectively. The gene expression profile chip technology was used to determine and analyze the changes in apoptosis-related gene expression after adherent culture and the expression of MDR1 mRNA was assessed by reverse transcriptional polymerase chain reaction (RT-PCR) at the same time.</p><p><b>RESULTS</b>In the adherent culture, the proliferation of the U937 cells was inhibited, the G0/G1 phase cells increased (F = 64.9726, P < 0.0001), G2/M phase cells were decreased (F = 98.1361, P < 0.0001) and the natural apoptosis rate was decreased (F = 24.0866, P < 0.0001) compared with those in the suspended culture. U937 cell viability was enhanced and cell apoptosis was blocked during DNR treatment in adherent culture with MSCs. Thirty-nine differently expressed genes were screened from the 487 apoptosis related genes in the adherent culture U937 cells. Among the 37 upregulated genes, Bcl-XL was upregulated most significantly. Two genes were downregulated. Adherent culture did not induce MDR1 mRNA expression in U937 cells.</p><p><b>CONCLUSIONS</b>MSCs play a role in modulating the proliferation of U937 cells and response of U937 cells to DNR, and Bcl-XL apoptosis-inhibiting gene may be most important in determining the sensitivity of leukemic cells to treatment, which is not related to MDR1.</p>

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR).</p><p><b>METHODS</b>The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant.</p><p><b>RESULTS</b>In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells.</p><p><b>CONCLUSIONS</b>shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.</p>

Effects of fluoride on osteoclastic activity of rats in vitro / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of fluoride on activities of tartrate-resistant acid phosphate (TRAP) and matrix metalloproteinase-9 (MMP-9) in rat osteoclasts cultured in vitro.</p><p><b>METHODS</b>Osteoclast was isolated mechanically from long bones of neonatal rats and cultured in vitro. Histochemical stain was applied to detect the effects of fluoride on activities of TRAP and in-situ hybridization was used to study the expression of MMP-9 mRNA in rat osteoclasts in vitro.</p><p><b>RESULTS</b>Number of TRAP positive cells was 154.2, 160.0, 170.6, 179.0 and 180.0 per cm(2), respectively for the rats with varied doses of fluoride, in a dose-response pattern but without statistical significance. The expression of MMP-9 mRNA increased with elevating dose of fluoride, especially in the rats with 1.00, 2.00 and 4.00 mg/L of fluoride, to 94.50, 94.64 and 104.97, respectively, significantly different from those in control group.</p><p><b>CONCLUSIONS</b>Fluoride can enhance the MMP-9 mRNA expression in cultured osteoclasts of rats.</p>