Study on the bone marrow mesenchymal stem cells induced drug resistance in the U937 cells and its mechanism.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia cells].

OBJECTIVE: To investigate the effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib. METHODS: Tumor suppressor gene p14(ARF) was transduced into K562 (K562-p14(ARF)) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control. Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency, and Western blotting assay was used to detect p14(ARF) protein of K562 cells. WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0, 0.015, 0.062, 0.125, 0.25, 0.5, 1.0, 2.0 µmol/L). Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively. RESULTS: Fluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14(ARF), K562-VSV and CML-BC1 cells were close to 100%, and CML-BC 2-4 cells were 80% to 90% on average. Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14(ARF) were significantly higher than of untransduced cells; the apoptosis rate of K562-p14(ARF) was 20%; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14(ARF) [(71.1±22.4)%] was significantly higher than of control group [(12.4±6.2)%] (P<0.05). Imatinib significantly inhibited the proliferation of K562-p14(ARF) cells in a dose-dependent manner. The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14(ARF) (41.5±13.2) was significantly lower than of the control group (88.5±7.9) (P<0.05). CONCLUSION: Increased p14(ARF) gene expression could induce apoptosis of CML cells; Moreover, it could enhance inhibitory effect on cell proliferation when combined with imatinib.

[Influence of gene transduction mediated by lentivirus vector on gene expression of human CD34+ cord blood cells].

OBJECTIVE: To investigate the influence of gene transduction mediated by lentivirus vector on human CD34+ cord blood cell (CBCs) gene expression. METHODS: CD34+ cells were isolated and transduced with the third-generation self-inactivating ( SIN) lentiviral vector carrying green fluorescent protein (GFP). The total RNA from transduced cells was extracted and the differences of genotypes between the transduced and non-transduced CD34+ cells were determined with cDNA microarray analysis. RESULTS: In 23000 genes two were upregulated and six downregulated. These changes were not confirmed by semi-quantitative RT-PCR method. CONCLUSIONS: Lentiviral vector used in this study do not influence significantly on the gene expression of CD34+ CBCs, and the vector system may be a useful and safe one in clinical gene therapy.

[Reversal of mdrl gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors].

OBJECTIVE: To explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM. METHODS: The oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM). RESULTS: In positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively. CONCLUSIONS: shRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.

[Study of apoptosis gene expression in U937 cells induced by adhesion culture with mesenchymal stem cell].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.