Sorting of side population cells from multiple myeloma cell lines and analysis of their biological characteristics / 中国实验血液学杂志

This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.

Effects of down-regulated TRAF6 gene expression on the proliferation and apoptosis in multiple myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells.</p><p><b>METHODS</b>Detection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot.</p><p><b>RESULTS</b>The levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein.</p><p><b>CONCLUSION</b>TRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.</p>

Effect of zoledronic acid on the growth and CD138 expression of myeloma cell line KM3 / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of zoledronic acid (ZA) on the growth and CD138 expression of myeloma cell line KM3.</p><p><b>METHODS</b>KM3 cells were treated with different concentrations of ZA The growth of KM3 cells was measured by trypan blue dye exclusion, and the changes of apoptosis rate, cell cycle and expression of CD138 induced by ZA by flow cytometry.</p><p><b>RESULTS</b>Within the concentration of 10(-5)-10(-3) mol/L, ZA obviously inhibited the growth of KM3 cells in a dose dependent manner. IBN at 10(-5)-10(-4) moL/L increased Annexin V positive rate, blocked cells at the S/G2 boundary, reduced the expression of CD138 and its fluorescence intensity.</p><p><b>CONCLUSION</b>ZA can inhibit the growth of KM3 cells in a dose-dependent manner and inhibited CD138 expression. The mechanism is probably related to induction cell cycle accumulation in S phase and apoptosis.</p>

Apoptosis induction and phosphorylated protein kinase C epsilon expression in 32D cells by sera from patients with aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of phosphorylated protein kinase C epsilon (pPKC epsilon) on apoptosis of 32D cells induced by sera from patients with aplastic anemia (AA).</p><p><b>METHODS</b>The expression of pPKC epsilon and apoptosis in 32D cells were measured by Western blotting and flow cytometry after incubation with sera from healthy individuals (controls, n = 8), patients with severe AA ( SAA, n = 8)and non severe AA (NSAA, n = 6).</p><p><b>RESULTS</b>After incubation for 0, 12, 24, 36 and 48 hours in the presence of serum and for another 4 hours in medium deprived of serum, the levels of pPKC epsilon in cells in SAA and NSAA group increased gradually, peaked at 24 hours, and then declined (P < 0.05). Compared with that in control group (0.54 +/- 0.08), pPKC epsilon was overexpressed in both SAA group (0.90 +/- 0.10) and NSAA group (0.64 +/- 0.08) (P < 0.05) after 24 hours incubation with serum and subsequent 4 hours without serum. pPKC epsilon level was higher in SAA group than in NSAA group (P < 0.05). A greater proportion of 32D cells showed apoptosis after 24 hours incubation with sera from SAA patients [(4.05 +/- 1.05)%] and subsequent 4 hours incubation without serum than that in controls [(2.45 +/- 0.51)%, P < 0.05], which was correlated with the same serum-induced expression of pPKC epsilon (r = 0.869, P < 0.05). Although the mean level of pPKC epsilon expression was higher in NSAA group than in control group, no significant difference of apoptosis was found between the two groups [(2.45 +/- 0.51)% vs (3.24 +/- 0.56)%, P > 0.05].</p><p><b>CONCLUSION</b>Sera from both SAA and NSAA patients could upregulate the expression of pPKC epsilon in 32D cells. The SAA sera induce apoptosis in 32D cells significantly, but the latter do not.</p>

Apoptosis of in vitro cultured BMMNC from MDS patients induced by arsenic sulfide / 中国实验血液学杂志

The aim of this study was to investigate the inhibition effect of arsenic sulfide (As2S2) on the growth of in vitro cultured BMMNC from MDS patients and to explore its possible cellular and molecular mechanisms. The apoptosis of MDS cells induced by As2S2 solution of different concentrations were studied with MTT, flow cytometry, and RT-PCR. The results showed that (1) low concentration of As2S2 (0-0.6 mg/L) had no marked inhibition effect on proliferation of MDS cells; (2) after treatment with 1.5-50 mg/L of As2S2, both low risk MDS cells and high risk MDS cells presented typical features of apoptosis with a dose-dependent manner, the expression of bcl-2 mRNA and the ratio of bcl-2/bax obviously decreased after As2S2 treatment (P < 0.05); (3) BMMNC from MDS patients had higher apoptosis ratio than that of BMMNC from control. It is concluded that BMMNC excessive apoptosis exists in MDS patients; low concentration of As2S2 (0-0.6 mg/L) shows no inhibition effect on proliferation of MDS cells; high concentration of As2S2 (1.5-50 mg/L) induces apoptosis of MDS cells.

Expression of vascular endothelial growth factor in patients with aplastic anemia and its significance / 中国实验血液学杂志

This study was aimed to investigate the expression of vascular endothelial growth factor (VEGF) in patients with aplastic anemia. The gene expressions of VEGF in mononuclear cells of bone marrow from 7 cases of aplastic anemia and 12 normal controls were detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The expressions of VEGF in bone marrow from 20 cases of aplastic anemia and 20 normal controls were also determined by immunohistochemistry assay. The results showed that expression of VEGF mRNA was found in 2 out of 7 (28.57%) bone marrow of patients and in 10 out of 12 (83.33%) bone marrow of normal controls. The VEGF mRNA in patients with aplastic anemia was significantly lower than that in normal controls (P < 0.05). No patients with aplastic anemia showed immunohistochemical staining of VEGF in bone marrow, while 5 out of 20 (25%) normal controls exhibited VEGF positive cells. Bone marrow of aplastic anemia patients contained less VEGF than that of normal persons (P < 0.05). In conclusion, when compared with normal controls, VEGF expression decreased significantly in patients with aplastic anemia at gene transcription level and protein translation level, it may be related to the defect of angiogenesis and thus hematopoiesis in bone marrow of patients with aplastic anemia.

Study on negative regulatory factors in bone marrow mononuclear cells of myelodysplastic syndromes / 中国实验血液学杂志

To explore the difference of negative regulatory factors among T lymphocyte subsets in bone marrow (BM) of myelodysplastic syndromes (MDS) and their relations to apoptotic gene Fas, different lymphocyte subsets in BM were categorized by monoclonal antibodies with 3 color fluorescence using flow cytometry, and the intracellular cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) were determined following marrow cells culture. Then Fas mRNA of bone marrow mononuclear cells (BMMNC) were examined by RT-PCR. The results showed that TNF-alpha, IFN-gamma levels in BM of MDS both increased, the former produced by cells CD4+CD45RO+, CD8+CD45RO+, the latter by cells CD4+CD45RO+, CD8+CD45RO+, CD8+CD45RA+, in which the cells CD8+CD45RO+ were dominant. Fas mRNA expression had relationship with IFN-gamma produced by T cells but not with TNF-alpha. It is concluded that in hematopoietic microenvironment of MDS, not only the T lymphocyte subsets are in disorder, but also negative regulatory factors secreted by T lymphocyte increase. T lymphocytes play an important role in producing IFN-gamma in patients with MDS.