NK4 inhibits the proliferation and induces apoptosis of human rheumatoid arthritis synovial cells

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by proliferation and insufficient apoptosis of synovial cells. NK4 is a hepatocyte growth factor antagonist and is implicated in cell proliferation, viability, and apoptosis of many tumour cells. This study aimed to investigate the role of NK4 in the regulation of human RA synovial cell proliferation and apoptosis. Fibroblast‐like synoviocytes (FLSs) isolated from RA patients and MH7A synovial cells were subjected to MTT, flow cytometry, and Western blot analysis. We found that NK4 suppressed cell proliferation through cell cycle arrest at the G0/G1 phase and induced apoptosis in RA synovial cells. Furthermore, NK4 altered the expression of cell cycle and apoptosis‐related proteins such as cyclin D1, cyclin B1, PCNA, p21, p53, Bcl‐2, Bax, cleaved caspase‐9, and cleaved caspase‐3. Additionally, NK4 reduced the phosphorylation level of NF‐κB p65 and upregulated the expression of sirt1, but did not change the levels of p38 and p‐p38 in RA‐FLS and MH7A cells. In conclusion, NK4 inhibits the proliferation and induces apoptosis of human RA synovial cells. NK4 is a promising therapeutic target for RA. We demonstrated that NK4 inhibited cell proliferation by inducing apoptosis and arresting cell cycle in RA‐FLS and MH7A cells. The apoptotic effects of NK4 may be mediated in part by decreasing Bcl‐2 protein level, increasing Bax and caspase 3 protein levels, and inhibiting NF‐κB signalling in RA‐FLS and MH7A cells. These findings reveal potential mechanism underlying the role of NK4 in RA synovial cells and suggest that NK4 is a promising agent for RA treatment.     

Expression pattern of cell cycle-related gene products in synovial stroma and synovial lining in active and quiescent stages of rheumatoid arthritis

OBJECTIVE: To investigate the expression pattern of cell cycle related gene products in active and quiescent Rheumatoid arthritis (RA). METHODS: Synovial tissue from 20 patients with active proliferative RA and 28 patients with RA in remission was immunohistochemically examined for expression of p53, p63, p21, p27, p16, cyclin D1, CDK4, RB, E2F, Ki-67 on tissue microarrays and by DNA flow cytometry for cell cycle phases. RESULTS: Elevated expression of p53 and p27 was found in synovial lining and in stromal cells in proliferative active RA. In the remission stage this finding was confined to the synovial lining. Most of the cells were in the G0-phase. Ki-67 proliferation index was maximum 10% in synovial cells. CONCLUSION: The p53 pathway is activated in synovial cells in active RA as well as in quiescent stage of disease. Differences in the spatial expression pattern of proteins involved in the p53 pathway in RA in remission compared to actively proliferating RA reflect the phasic nature of the disease and support in our opinion the concept of adaptive role of p53 pathway in RA.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

α-烯醇化酶基因干扰表达对籽鹅卵泡颗粒细胞增殖及凋亡的影响*

目的: 研究α-烯醇化酶(ENO1)基因干扰表达对籽鹅卵泡颗粒细胞增殖、凋亡和细胞周期的影响。方法: 原代培养籽鹅F1级卵泡颗粒细胞(复合培养),将ENO1基因干扰表达重组质粒转染至籽鹅卵泡颗粒细胞。实验分为四组:ENO1干扰表达组(RNAi)、无关序列干扰组(NC)、培养液组(Control)、转染试剂组(Lip)。流式细胞术检测干扰组和对照各组的凋亡率、细胞周期时相性。结果: ENO1基因干扰表达使籽鹅卵泡颗粒细胞增殖速度变慢,凋亡率增加,G2/M期颗粒细胞比例增高。结论: ENO1基因干扰表达可使籽鹅卵泡颗粒细胞周期发生G2/M期阻滞,诱导细胞发生凋亡,抑制细胞增殖。     

Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 μM) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cyclin D3 and c-MYC control glucocorticoid-induced cell cycle arrest but not apoptosis in lymphoblastic leukemia cells

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells. To investigate cell cycle effects of GC in the absence of obscuring apoptotic events, we used human CCRF-CEM leukemia cells protected from cell death by transgenic bcl-2. GC treatment arrested these cells in the G1 phase of the cell cycle due to repression of cyclin D3 and c-myc. Cyclin E and Cdk2 protein levels remained high, but the kinase complex was inactive due to increased levels of bound p27(Kip1). Conditional expression of cyclin D3 and/or c-myc was sufficient to prevent GC-induced G1 arrest and p27(Kip1) accumulation but, importantly, did not interfere with the induction of apoptosis. The combined data suggest that repression of both, c-myc and cyclin D3, is necessary to arrest human leukemia cells in the G1 phase of the cell division cycle, but that neither one is required for GC-induced apoptosis.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells

We studied the effects of apigenin on the cell cycle distribution and apoptosis of human breast cancer cells and explored the mechanisms underlying these effects. We first investigated the antiproliferative effects in SK-BR-3 cells exposed to between 1 and 100 microM apigenin for 24, 48 and 72 h. Apigenin significantly inhibited cell proliferation at concentrations over 50 microM, regardless of exposure time (P<.05), and resulted in significant cell cycle arrest in the G(2)/M phase after 48 h of treatment at high concentrations (50 and 100 microM; P<.05). To investigate the regulatory proteins of cell cycle arrest affected by apigenin, we treated cells with 50 and 100 microM apigenin for 72 h. Apigenin caused a slight decrease in cyclin D and cyclin E expression, with no change in CDK2 and CDK4. In addition, the apigenin-induced accumulation of the cell population in the G(2)/M phase resulted in a decrease in CDK1 together with cyclin A and cyclin B. In an additional study, apigenin also increased the accumulation of p53 and further enhanced the level of p21(Cip1), with no change in p27(Kip1). The expression of Bax and cytochrome c of p53 downstream target was increased markedly at high concentration treatment over 50 microM apigenin. Based on our findings, the mechanism by which apigenin causes cell cycle arrest via the regulation of CDK1 and p21(Cip1) and induction of apoptosis seems to be involved in the p53-dependent pathway.     

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.     

Regulation of survivin by retinoic acid and its role in paclitaxel-mediated cytotoxicity in MCF-7 breast cancer cells

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.     

CSE1L/CAS, a microtubule-associated protein, inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs

CSE1L/CAS, a microtubule-associated, cellular apoptosis susceptibility protein, is highly expressed in various cancers. Microtubules are the target of paclitaxel-induced apoptosis. We studied the effects of increased or reduced CAS expression on cancer cell apoptosis induced by chemotherapeutic drugs including paclitaxel. Our results showed that CAS overexpression enhanced apoptosis induced by doxorubicin, 5-fluorouracil, cisplatin, and tamoxifen, but inhibited paclitaxel-induced apoptosis of cancer cells. Reductions in CAS produced opposite results. CAS overexpression enhanced p53 accumulation induced by doxorubicin, 5-fluorouracil, cisplatin, tamoxifen, and etoposide. CAS was associated with alpha-tubulin and beta-tubulin and enhanced the association between alpha-tubulin and beta-tubulin. Paclitaxel can induce G2/M phase cell cycle arrest and microtubule aster formation during apoptosis induction, but CAS overexpression reduced paclitaxel-induced G2/M phase cell cycle arrest and microtubule aster formation. Our results indicate that CAS may play an important role in regulating the cytotoxicities of chemotherapeutic drugs used in cancer chemotherapy against cancer cells.     

LRP16影响宫颈癌Siha细胞对化疗药物的敏感性

目的:探讨抑制LRP16的表达对宫颈癌Siha细胞的化疗药物敏感性的影响。方法:将抑制LRP16表达的小干扰RNA:negativecontrol-si RNA(NC)、si RNA-374(si374)转染入Siha宫颈鳞癌细胞系中,通过顺铂(DDP)和紫杉醇(TAX)的处理后,采用CCK-8检测不同浓度紫杉醇、顺铂作用宫颈癌细胞系Siha48 h后,计算出细胞被抑制一半时顺铂、紫杉醇的药物浓度(IC50);使用Hoechst33342染色观察细胞凋亡,采用流式细胞仪检测顺铂IC50作用Siha细胞48小时后的细胞凋亡情况,紫杉醇IC50作用Siha细胞之后的细胞周期分布情况。结果:CCK-8检测转染的Siha细胞增殖活性受到抑制,Hoechst33342染色观察转染的Siha细胞凋亡明显增加,流式细胞仪检测凋亡显示,si374+顺铂的早期凋亡率22.15±2.24,NC+顺铂12.45±2.72,流式细胞仪检测周期显示G2/M(%),si374+紫杉醇29.94±1.87,NC+紫杉醇17.66±2.32。结论:LRP16基因表达下调之后,抑制Siha细胞的增殖、促进其凋亡,使细胞周期滞留于G2/M期,从而提高Siha细胞的化疗敏感性。     

Effects of 2-methoxyestradiol on proliferation, apoptosis and gene expression of cyclin B1 and c-Myc in esophageal carcinoma EC9706 cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-estradiol. In this study, we determined the antitumour activities of 2-ME on the well-differentiated EC9706 esophageal carcinoma cells in vitro. 2-ME had a strong antiproliferative effect on EC9706 cells and caused an increase in the population of apoptotic cells, detected by flow cytometry. A significant number of cells were blocked in the G(2)/M phase of the cell cycle. 2-ME-treated cells demonstrated an increase in cyclin B1 and c-Myc protein levels, as well as an increase in the percentage of G(2)/M phase. Their up-regulation may be involved in 2-ME-induced apoptosis and G(2)/M cell cycle arrest of the EC9706 cells, and it precedes the onset of apoptosis.     

RNA interference-mediated silencing of the PAR gene inhibits the growth of PC3 cells via the induction of G2/M cell cycle arrest and apoptosis

BACKGROUND: The prostate androgen-regulated (PAR) gene is ubiquitously overexpressed in prostate cancer (PCa) cells and is involved in proliferation of PCa. However, the mechanism by which the modulation of PAR gene expression elicits its biological effects on PCa cells is not well documented. Here, we investigate the mechanism of PAR depletion inhibiting PCa cell growth. METHODS: PAR expression was depleted by small interfering RNA (siRNA) and its subsequent effects on proliferation of PC3 cells were determined by the trypan blue exclusion assay. Flow cytometric analysis provided the evidence for the progression of cell cycle and the induction of apoptosis which was further confirmed by the observation of cleavage of poly(ADP-ribose) polymerase. Western blot analysis was performed to investigate the involvement of critical molecular events known to regulate the cell cycle and the apoptotic machinery. RESULTS: siRNA transfection results in a dose-dependent inhibition of cell growth in PC3 cells by causing G2/M phase cell cycle arrest and apoptosis. The G2/M arrest by PAR depletion was associated with decreased levels of cyclin B1, pCdc2 (Tyr15), Cdc2 and Cdc25C. PAR depletion also was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax : Bcl-2. CONCLUSIONS: Our data indicate that PAR depletion induces G2/M arrest via the Cdc25C-Cdc2/cyclin B1 pathway. Furthermore, the results of the present study point toward involvement of pathways mediated by both caspase 8 and caspase 9 in apoptosis induction by PAR depletion.     

Digital Holographic Microscopy for Non-Invasive Monitoring of Cell Cycle Arrest in L929 Cells

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Cyclic AMP delays G2 progression and prevents efficient accumulation of cyclin B1 proteins in mouse macrophage cells

In mouse macrophage cells, the increase of the intracellular cAMP level activates protein kinase A (PKA) and results in inhibition of cell cycle progression in both G1 and G2/M phases. G1 arrest is mediated by a cdk inhibitor, p27Kip1, which prevents G1 cyclin/cdk complexes from being activated in response to colony stimulating factor-1, whereas inhibition of G2/M progression has not been fully elucidated. In this report we analyzed the effect of cAMP on G2/M progression in a mouse macrophage cell line, BAC1.2F5A. Flow cytometric analysis and mitotic index measurement using both synchronized and asynchronized cells revealed that addition of cAMP-elevating agents (8-bromoadenosine 3':5'-cyclic monophosphate and 3-isobutyl-methyl-xanthine), although they did not affect S phase progression or M/G1 transition, temporarily arrested cells in G2 but eventually the cells proceeded to M phase, resulting in about 4 hours delay of G2 progression. Timing of cyclin B1/Cdc2 kinase activation was also retarded by about 4 hours, which was accompanied by inhibition of efficient accumulation of cyclin B1 proteins. Initial induction and accumulation of cyclin B1 mRNA were not hampered, but the half life of cyclin B1 proteins was significantly shorter during G2 phase in the presence of cAMP-elevating agents compared with that of the cells blocked from progressing through M phase by nocodazole. These results imply that the cAMP/PKA pathway regulates G2 phase progression by altering the stability of a crucial cell cycle regulator.     

Helicobacter pylori γ-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition

In our previous study, we showed that Helicobacter pylori γ-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclindependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.     

MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth-retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were cultured in the presence of SF. In contrast, GM-CSF in SFs existed at a significant level in the patients with RA (n = 6), in comparison with the other inflammatory cytokines, IL-1beta and TNF-alpha. Most RA FLSs at passage 10 or more recovered from their growth retardation when cultured in the presence of SF. The SF-mediated growth recovery was markedly impaired by anti-GM-CSF antibody. Growth-retarded RA FLSs recovered their proliferative capacity after treatment with GM-CSF in a dose-dependent manner. However, MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation of RA FLSs. Taken together, our results imply that MLN51, induced by GM-CSF, is important in the proliferation of RA FLSs in the pathogenesis of RA.