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Objective: To study the regulation of the proliferation of epiphysis stem cells by the PTHrP (parathyroid hormone related peptide) and Notch signaling systems., Methods: An organ culture system of femurs of SD rat in 24 h after birth was employed. PTHrP (1 - 34) was used as the activator of the PTHrP signaling pathway and PTHrP (7 - 34) as the antagonist of PTH (parathyroid hormone)-receptor. For Notch signaling system, Jagged1/Fc was used as the activator and DAPT as its inhibitor. The femurs were cultured in DMEM (Dulbecco's modified Eagle's medium)/F12 medium while phosphate buffered saline was used for the control groups. Hematoxylin and eosin staining and bromodeoxyuridine analysis were used to analyze the length of the epiphysis stem cells zone and the proliferation of epiphysis stem cells. The expression of NICD (Notch intra-cellular domain) and Jagged1 were analyzed by immunohistochemistry. The epiphysis stem cells were transfected with the lentiviral vectors with rat PTHrP gene overexpression or inhibition properties, the cells transfected with the PGC-GFP-lentivirus or NC-GFP-lentivirus were used as control. Western blot was employed to detect the expression of NICD and Jagged1 genes., Results: PTHrP (1 - 34) and Jagged1/Fc could dramatically elevate the rate of epiphysis stem cells zone by the whole growth plate length measurement while PTHrP (7 - 34) and DAPT could decrease the rate. Brdu analysis also showed that the number of proliferative epiphysis stem cells could be up-regulated by the PTHrP (1 - 34) or Jagged1/Fc signaling. By contrast, the treatment with PTHrP (7 - 34) or DAPT reduced the number of proliferative epiphysis stem cells. Immunohistochemistry and Western blot showed a significantly elevated expression of NICD and Jagged1 when PTHrP signaling was activated while a reductive expression of NICD and Jagged1 when PTHrP signaling was inactivated., Conclusion: Both of PTHrP and Notch signaling system could promote the proliferation of epiphysis stem cells. And the PTHrP signaling can stimulate Notch signaling to promote the proliferation of epiphysis stem cells.

Background: Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis., Methods: Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes., Results: In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runx1 cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runx1 group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months., Conclusions: Overexpression or knock-down of Runx1 gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runx1 expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo.

Objective: To investigate the effects of receptor-interacting protein (RIP)140 gene knockdown on the proliferation of microglioma cells., Methods: Mouse microglioma cells of the line BV-2 were cultured and transfected with 2 kinds of recombinant RIP140-shRNA plasmids (V2MM-71674 and V2MM-71080) or blank plasmid MSCV-EGFP. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of RIP140; and the cell proliferation was detected by MTT assay., Results: There were not significant differences in the RIP140 mRNA and protein expression between the BV-2 and BV-2-MGCV-EGFP groups. Compared to those of the BV-2 group, the RIP140 mRNA expression levels of the BV-2-71674 and BV-2-71080 groups were lower by 73% and 75% respectively. The protein expression levels of the BV-2-71674 and BV-2-71080 groups were remarkably lower than those of the BV-2 and BV-MSGV-EGFP groups. MTT assay showed that there were not significant differences in the proliferation rates at different time points between the BV-2 and BV-2-MSCV-EGFP groups, however, the proliferation rates at the time points of 24, 48, 72, and 96 h of the BV-2-71674 and BV-2-71080 groups were significantly lower than those of the BV-2 group (all P<0.01)., Conclusion: RIP140 gene knockdown effectively inhibits the proliferation of microglioma cells.

Background & Objective: Myelodysplastic syndromes (MDS) are heterogeneous disorders with the expansion of malignant clones. No satisfactory treatment for MDS is available. Sodium valproate (VPA) can inhibit the proliferation of tumor cells by inducing G(0)/G(1) phase arrest and cell apoptosis. This study was to investigate the effects of VPA on the proliferation of MDS cell line MUTZ-1, and explore possible mechanisms., Methods: MUTZ-1 cells were treated with VPA. Cell proliferation was determined by MTT assay. Cell morphology was observed under microscope and transmission electron microscope. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). The expression of p21(WAF1) (cyclin-dependent kinase inhibitor) was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot., Results: VPA inhibited the proliferation of MUTZ-1 cells in concentration-and time-dependent manners. When treated with 4 mmol/L VPA for 72 h, typical apoptotic morphologic features appeared in MUTZ-1 cells: condensation of cells and nuclear chromatin, disintegration of nuclear chromatin, and apoptotic bodies were observed under microscope; aggregation and margination of apoptotic nuclear chromatin, cytoplasm condensation, and irregular chromatin masses were observed under transmission electron microscope. The apoptosis rate was significantly higher in the cells treated with 1, 2, and 4 mmol/L VPA for 72 h than in untreated cells [(3.14+/-0.87)%, (14.90+/-1.04)% and (22.46+/-1.74)% vs. (0.99+/-0.35)%, P<0.05]. After treatment of VPA, cell cycle was arrested obviously at G0/G1 phase (P<0.05)û the mRNA and protein levels of p21(WAF1) were up-regulated in MUTZ-1 cells (P<0.05)., Conclusions: VPA could induce G(0)/G(1) phase arrest, inhibit the proliferation and induce the apoptosis of MUTZ-1 cells in vitro. The mechanism may be associated with the up-regulation of p21(WAF1).