Effects of human cytomegalovirus on the cell cycle of duct epithelial cell cultures of human salivary gland in vitro and relative mechanism / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the effects of human cytomegalovirus (HCMV) on the cell cycle of duct epithelial cell cultures of human salivary gland (HSG) in vitro and relative mechanism.</p><p><b>METHODS</b>HSG was cultured in vitro. Reverse transcriptase polymerase chain reaction (RT-PCR) and nest-RT-PCR were used respectively to investigate ie1/ie2 transcription in HSG infected by human cytomegalovirus(HCMV). The effects of HCMV on the cell cycle of HSG were studied by flow cytometry in vitro. The expression of cyclin D1 in HSG infected by HCMV was detected by Western blotting.</p><p><b>RESULTS</b>HCMV iel/ie2 transcription could be detected in HSG infected by HCMV. HCMV arrested productively infected cells in G1 stage. And cyclin D1 was down-regulated in HCMV infected HSG.</p><p><b>CONCLUSION</b>HCMV inhibits proliferation of HSG by affecting G1/S check point and down-regulating cyclin D1 in vitro.</p>

Effect of curcumin in combination with bortezomib on proliferation and apoptosis of human multiple myeloma cell line H929 and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the effect of curcumin in combination with bortezomib on the proliferation and apoptosis of human MM cell line H929 in vitro, and to explore its mechanisms. MTT assay was applied to detect the inhibitory effects of curcumin and bortezomib either alone or combined at different concentrations on H929 cells, and flow cytometry was employed to assay the apoptosis rate. In addition, RT-PCR was used to analyze the mRNA expression of gene BCL-2, BAX, cyclin D1. Immunofluorescence technique was performed to study the location changes of NF-κB P65 in different groups. The results showed that both curcumin and bortezomib inhibited the proliferation of MM cell line H929 in dose-dependent manner, and combination of these two drugs displayed synergistical effect. A much higher apoptosis rate was determined by flow cytometry in combinative groups than that in single or control group. And RT-PCR showed, as compared with curcumin or bortezomib group, there was mRNA expression decrease of BCL-2, cyclin D1 but increase of BAX in combined group. The expression of NF-κB P65 in nucleus was downregulated in either the curcumin or bortezomib group, however, distribution of NF-κB P65 in cytoplasm was observed in combined group. It is concluded that the combination of curcumin and bortezomib is much more effective for the inhibiting proliferation and promoting apoptosis of H929 cell line, which may function by inhibiting the transcription of NF-κB and apoptosis-related genes.

Bortezomib depresses osteoblast apoptosis induced by mouse myeloma cells / 中国实验血液学杂志

The purpose of this study was to explore the effect of proteasome inhibitor, bortezomib (Bzb), on osteoblast in pathologic status of myeloma bone disease. The myeloma bone disease was modeled by co-culture of mouse myeloma cell RPMI8226 with osteoblast line MC-3T3E1 from mouse calvaria, and intervenient culture of supernatant. The inhibitory effect of Bzb on proliferation of MC-3T3E1 assayed by modified MTT method, the apoptosis of MC-3T3E1 cells was determined by flow cytometry with Annexin V/PI staining, the expressions of osteoblast markers, Runx2/cbfa1, osteocalcin (OCN) and osterix (OSX) in MC-3T3E1 treated with Bzb were detected by RT-PCR and Western blot respectively. Experiments were divided into 3 group: single cultured, co-cultured and supernatant-interveniently cultured groups. The results showed the Bzb in higher concentration inhibited proliferation of MC-3T3E1 cells in a dose-dependent manner, with the IC(50) of 38.1 nmol/L for 48 hours, the Bzb in low concentration (5 nmol/L) did not show the inhibitory effect on proliferation of MC-3T3E1 in single cultured group (p>0.10), but could decrease apoptotic rate of MC-3T3E1 by 32.5% and 24.6% respectively in cocultured and supernatant-interveniently cultured groups, moreover increased the expression of osteoblast-related gene OSX, OCN mRNA and protein (p<0.05), while no obvious change of Runx2/cbfa1 expression was observed (p>0.05). It is concluded that the proteasome inhibitor, Bzb, in low concentration promotes the activity of osteoblast internal mechanisms, and prevents the apoptosis of osteoblasts induced by myeloma cells. In addition, it can up-regulate transcription and expression of osteoblast markers related to Runx2/cbfa1 path way, thus may protect osteoblasts in myeloma bone disease.

Bcl-6 expression in K562 cells and its role in mechanism underlying induced differentiation into various myelocytic lineages / 中国实验血液学杂志

This study was purposed to investigate the changes of bcl-6 expression in K562 cells and the mechanism inducing differentiation into different myelocyte lineages. Models of K562 cells inducing differentiation to lineages of megakaryocyte, erythrocyte and macrophagocyte were established with inducers TPA (tetradecanoylphorbol 13-acetate), Hu (hydroxyurea) and HMBA (hexamethylene bisacetamide) respectively. Western blot assay was applied to detect the expression of bcl-6 in K562 cells before and after the induction. Meanwhile, PCR, cloning and direct DNA sequencing were used to identify mutations in the 5' regulatory region of bcl-6 in K562 cells before and after induction with TPA. The results indicated that up-regulation of bcl-6 expression was found only in K562 cells being induced differentiating into megakaryocyte-lineage, while mutation of 5' regulatory region of bcl-6 gene was not found. It is concluded that expression of bcl-6 increases only when K562 cells differentiate into megakaryocyte lineage and bcl-6 expression may play an important role in K562 cells induced differentiation into megakaryocyte lineage. The up-regulation of bcl-6 expression may not be related with the mutation of 5' regulatory regions of the gene.

Apoptosis of multiple myeloma cells induced by gossypol acetic acid in vitro and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the apoptosis effect of gossypol acetic acid on classic human multiple myeloma RPMI8226 cell line in vitro and its mechanism. The inhibitory effect on proliferation of RPMI8226 cells was evaluated by means of MTT assay. Cytotoxic effect and apoptosis was identified and analyzed with the aid of transmission electron microscopy, mitochondria membrane potential (MMP) and DNA gel electrophoresis. Meanwhile, Western-blot assay was used to detect the changes of several key cell apoptosis regulatory proteins such as BAX, caspase-3 and caspase-8 in these cells before and after treatment. The results showed that low concentrations of gossypol acetic acid (> 16 micromol/L) could suppress the proliferation and induce the apoptosis in RPMI8226 cells effectively. At the same time, gossypol acetic acid could also down-regulate the mitochondrial membrane potential, up-regulate the expression of the apoptosis-related protein such as BAX and caspase-3. It is concluded that the gossypol acetic acid can selectively induce proliferation inhibition and apoptosis of multiple myeloma RPMI8226 cells with a smaller dose.

Changes and mechanism of apoptosis-related gene expression in T lymphocytic leukemia JM cells induced with matrine / 中国实验血液学杂志

This study was purposed to investigate the changes of apoptosis-related gene expression in T lymphocytic leukemia JM cells induced with matrine, and its possible mechanism. JM cells was induced with 0.4 mg/ml matrine for 4 days, the total RNA was extracted from JM cells before and after matrine induction, the differential expression of apoptosis-related genes were screened with cDNA Expression Array Kit, the expression change of a part of gene was checked by Western blot. The results indicated that after induction of JM cells with matrine, differential expression of 31 genes were found by gene chip hybridization, the expression of caspase 8 was up-regulated more than 5 times. Western blot analysis showed that the up-regulation of caspase 8 gene expression positively correlated with induction time. It is concluded that differential expressions of many apoptosis-related genes in JM cells can be induced by matrine, in which gene expression of caspase 8 is up-regulated notably.

Differentiation of HL-60 cells directly cocultured with HFCL cells and alteration of their gene expression profile / 中国实验血液学杂志

To study the molecular mechanism of the effect of fibroblastoid stromal cells (HFCL) from human bone marrow on the proliferation and differentiation in acute myeloid leukemia HL-60 cells, the cell cycle was analyzed by flow cytometry (FCM); the cell differentiation was determined by morphology NBT test and flow cytometric detection for expression of CD11b, CD14, CD13 and CD33; the genes differently expressed between HL-60 cells and HL-60 cells directly cocultured with HFCL were detected by using Affymetric oligo microarray technique. The changes of expression in some key genes were confirmed by using RT-PCR and Northern blot. The results showed that the percentage of G(1) phase cells in AML cells cocultured with HFCL cells was higher than that without HFCL cells, and the percentage of S phase cells was lower. The NBT positive cells and the expression of CD11b and CD14 increased. It was found that after direct contact of HL-60 cells with HFCL cells for 96 hours, the expression levels of 582 genes were up-regulated, 1 323 genes down-regulated. It is concluded that many genes may take part in the influence of HFCL cells on HL-60 cells, which may give important insights into the important molecules and pathways or cross-talk involved in the interaction between the AML cells and stromal cells.

Difference of gene expression profiles between HL-60/VCR and HL-60 cells detected by human genome genechip / 中国实验血液学杂志

This study was aimed to detect the gene expression profile changes between human acute leukemia cell line HL-60 and VCR-resistance HL-60, and to investigate the underlying mechanisms of MDR by using genechip technology. In experiments, mRNA were harvested using TrizoL reagent from these two cell lines, through RT-PCR, the biotinylated nucleotide were incorporated into the cRNA during the in vitro transcription reaction. The high quality RNA was hybridized to the gene expression array--human genome U133A developed by Affymetrix. It was scanned by G2500A GeneArray Scanner and the acquired image was analysed by a series of softwares. The results showed that 5,507 genes were differentially expressed between human acute leukemia cell line HL-60 and VCR-resistant HL-60. Compared with HL-60, 3,100 genes were up-regulated and 2,407 genes were down-regulated in VCR-resistant cell line. These genes were involved in different cell activities such as growth regulation and signal transduction. Among the genes with remarkable differential expression between the two cell lines, 435 were up-regulated and 605 were down-regulated. It is concluded that many different kinds of genes are involved in the mechanism of MDR and there is an intricate molecular network that controls the sensitivity of leukemia cells to the chemotherapeutic agents. Genechip is an efficient tool for parallel gene expression analysis.

Rac1 expression and its effects on the cell cycle progression and apoptosis in human acute leukemic cell line HL-60 / 中国实验血液学杂志

The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.

Effects of HCMV on phenotypes of parotid duct epithelial cells and its mechanisms / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the effects of HCMV infection on phenotypes of parotid duct epithelial cells and relative mechanisms.</p><p><b>METHODS</b>The expressions of immediate early antigen of HCMV, pan cytokeratin and cathepsin D etc. were detected by immunohistochemical staining in tissues of parotid cytomegalic inclusion disease.</p><p><b>RESULTS</b>Cytokeratin which acts as an epithelial marker became negative while staining of Cathepsin D was intensified in parotid duct epithelial cells after infected by HCMV.</p><p><b>CONCLUSION</b>It demonstrated that cytokeratin was lost through over-expression of Cathepsin D in parotid duct epithelial cells infected by HCMV.</p>

Effect of bone marrow stromal cells on the apoptotic sensitivity of HL-60 and HL-60/VCR cells / 中国实验血液学杂志

This study was aimed to investigate the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on chemosensitivity of acute myeloid leukemia sensitive HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cell line in vitro co-culture. Setting up co-culture system of HL-60 or HL-60/VCR cells in direct contact with HFCL cells, or with HFCL cells separated by transwell, and exposing HL-60 or HL-60/VCR cells to different concentrations of topotecon (TPT), morphologic evidence for apoptosis was determined by staining with Wright-Giemsa stain and acridine orange/ethidium bromide (AO/EB). Cell cycle, sub-G(1) and annexin V FITC staining were detected by flow cytometry. The expression of active caspase-3, Bcl-2 and Pgp was detected by Western blot. The results showed that HL-60 or HL-60/VCR cells treated by TPT revealed characteristic apoptotic morphological changes by Wright-Giemsa and AO/EB staining. The percentage of annexin V-positive cells and apoptotic cells decreased when they were cocultured with HFCL cells. The proportion of G(0)/G(1) HL-60 or HL-60/VCR cells treated by TPT increased and the sub-G(1) appeared significantly, but apoptotic and sub-G cells reduced after direct contact with HFCL cells. Meanwhile, although HL-60 or HL-60/VCR cells treated by TPT expressed activated caspase-3, and the expression of Bcl-2 decreased, the expression of activated caspase-3 decreased and Bcl-2 increased after direct contact with HFCL cells. In conclusion, HFCL stromal cells can prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active caspase-3.

Role of Bcl-xL in the cathepsin D-associated apoptosis of K562 cells / 中国实验血液学杂志

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.

Human cytomegalovirus inhibits proliferation of duct epithelial cells of human salivary gland / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of human cytomegalovirus (HCMV) on the proliferation of duct epithelial cells of human salivary gland (HSG).</p><p><b>METHODS</b>The expression of proliferating cell nuclear antigen (PCNA) and p53 were studied in 11 cases of parotid cytomegalic inclusive disease (PCID) using immunohistochemical staining method. The effects of human cytomegalovirus (HCMV) on the proliferation of HSG were investigated by MTT method in vitro. The expression of PCNA in HSG infected by HCMV was examined using immunocytochemical staining and Western blotting.</p><p><b>RESULTS</b>PCNA was expressed weakly in most of megalic inclusion cells which were positive for HCMV, while all the megalic inclusion cells were p53 negative in all 11 cases of PCID. HCMV inhibited proliferation of HSG in vitro in a time dependent and dose dependent manner. Down-regulation of PCNA was shown in infected cells.</p><p><b>CONCLUSION</b>HCMV inhibits proliferation of HSG and down-regulation of PCNA may be an expression of the inhibition.</p>

Effects of normal human bone marrow fibroblastoid stromal cell line on the proliferation of multiple myeloma cell line RPMI8226 / 中国实验血液学杂志

To investigate the effects of normal human bone marrow fibroblastoid stromal cell lines HFCL on the proliferation of multiple myeloma cell lines RPMI8226, the co-culture system of RPMI8226 with HFCL was established, the adhesion ratio was determined by MTT colorimetric assay, growth curves were determined by trypan blue exclusion, the mitotic index (MI) of RPMI8226 cell was observed by Wright-Giemsa staining. Flow cytometry and Western blot were used to study the changes of cell cycle and expression of proliferating cell nuclear antigen (PCNA), respectively. The results showed that in co-culture the adhesion ratio of RPMI8226 after 1 hour was 29.4%; the proliferation of RPMI8226 cell line in direct contact with HFCL cell line was inhibited as compared with RPMI8226 in suspension. No obvious changes were observed in RPMI8226 cell separated by transwell inserts. The percentage of G(1) phase cells of RPMI8226 cell line in direct contact with HFCL was higher than that of RPMI8226 in suspension, and the percentage of S phase cells was lower. Moreover, the MI of RPMI8226 cell line in suspension was higher than that of RPMI8226 cell line in direct contact with HFCL cell. The expression of PCNA in RPMI8226 cell line in suspension was higher than that of RPMI8226 cell in direct contact with HFCL cell. It is concluded that the normal human bone marrow fibroblastoid stromal cell HFCL inhibits the proliferation and progression of cell cycle of multiple myeloma cell line, RPMI8226.

Matrine effects on JM cells by inhibiting proliferation and inducing apoptosis / 中国中药杂志

<p><b>OBJECTIVE</b>To study effects of matrine on JM cell strain.</p><p><b>METHOD</b>Morphologic changes were observed under light microscope with Wright-Giemsa staining, fluorescence microscope with Hoechst 33,258 staining and electron microscope. Alteration of cell cycle of different dose treating groups at the fourth day and 0.8 mg.mL-1 treatment group at the first, second, third, fourth day was analyzed by Flow cytometry. DNA ladder was detected with gel electrophoresis.</p><p><b>RESULT</b>From the third day after treatment of matrine, typical apoptosis features of cells were observed under light microscope and electron microscope in all test groups, and the features were more prominent with the time prolonging. At fourth day, flow cytometry analysis showed that there were sub-G1 peaks in all groups. From 0.1, 0.2, 0.4, 0.6 to 0.8 g.L-1 treatment groups, the rate of apoptotic cells to total cells were 3.1%, 2. 5%, 13.3%, 40.4%, 48.6%, respectively, and what in the control group was 1.4%; the rate of S phase cells to total cells was 28.9%, 26.1%, 27.7%, 0.9%, 14.2%, what in the control group was 30.4%; the rate of G1 phase cells to total cells was 63. 2%, 67.5%, 68.1%, 75.2%, 83.6%, what in the control group was 41.8%; From the first, second, third to fourth day, the rate of apoptotic cells to total cells of 0.8 mg.mL-1 treatment group were 3.0%, 3.7%, 9.1%, 48.6%, respectively; the rate of S phase cells to total cells was 28.6%, 17.5%, 19.1%, 14.2%; the rate of G1 phase cells to total cells were 45.5%, 77.3%, 77.2%, 83.6%. Gel electrophoresis displayed "DNA ladder" in 0.4, 0.6, 0.8 g.L-1 groups, while 0.1 and 0.2 g.L-1 groups didn't show such result.</p><p><b>CONCLUSION</b>Matrine can repress DNA synthesis and arrest JM cell strain at G1 phase, sequentially inhibiting the proliferation of the cell. Besides, this alkaloid can induce the apoptosis of JM cells.</p>

Effects of human fibroblastoid stromal cell line on proliferation of HL-60 cells and expression of VEGF / 中国实验血液学杂志

To investigate the effects of normal human bone m arrow fibroblastoid stromal cell line (HFCL) on the proliferation of acute myeloid leukemia cell line HL-60 and expression of vascular endothelial growth factor (VEGF), establishing coculture system of leukemia cell line HL-60 and HFCL, growth data was obtained by cell counting. Mitotic index (MI) was observed under Wright-Giemsa staining. Flow cytometry and Western blot were used as assays for cell cycle and expression of proliferating cell nuclear antigen (PCNA) separately. VE GF levels were evaluated by using commercial ELISA kits. The results showed that compared with HL-60 cells without HFCL cells, the proliferation of HL-60 cells in direct contact with HFCL cells and with HFCL cells separated by transwell was inhibited. The MI of HL-60 cells without HFCL cells was highest followed by HL-60 cells separated by transwell and HL-60 cells in direct contact with HFCL cells. The expression of PCNA in HL-60 cells with HFCL cells were lower than HL-60 cells without HFCL cells. Meanwhile, the percentage of HL-60 cells in G1 phase cocultured with HFCL cells was higher than that without HFCL cells while the percentage of Sphase cells was lower. The levels of VEGF in HL-60 cells with HFCL cells were lower than that in HL-60 cells alone. In conclusion, the normal bone marrow fibroblastoid stromal cells inhibited the proliferation of HL-60 cells as well as the expression of VEGF.

Inclusion bodies of human cytomegalovirus are composed of the DNA and immediately early and early antigens of the virus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the composition and significance of the inclusion bodies of human cytomegalovirus (HCMV).</p><p><b>METHODS</b>Microdissection of inclusion bodies, PCR and Southern blot were adopted to detect DNA, and immunohistochemistry method and catalyzed signal amplification (CSA) were used to detect the different antigens of HCMV.</p><p><b>RESULTS</b>The inclusion bodies of HCMV were separated from the tissue section of human salivary gland. The fragments amplified by PCR from these dissected inclusion bodies were confirmed to be the DNA of HCMV. With the immunohistochemical method CSA, the immediately early and early antigens of HCMV were detected with monoclonal antibodies DDG9/CCH2, while matrix protein AAC10 was negative in the inclusion bodies.</p><p><b>CONCLUSION</b>The ingredient of inclusion bodies of HCMV included the DNA and the antigens expressed in specific stage of the virus.</p>