Rab20 is critical for bacterial lipoprotein tolerization-enhanced bactericidal activity in macrophages during bacterial infection.

Bacterial cell wall component-induced tolerance represents an important protective mechanism during microbial infection. Tolerance induced by the TLR2 agonist bacterial lipoprotein (BLP) has been shown to attenuate the inflammatory response, and simultaneously to augment antimicrobial function, thereby conferring its protection against microbial sepsis. However, the underlying mechanism by which BLP tolerance augments bactericidal activity has not been fully elucidated. Here, we reported that the induction of BLP tolerance in murine macrophages upregulated the expression of Rab20, a membrane trafficking regulator, at both the mRNA and protein levels upon bacterial infection. The knockdown of Rab20 with Rab20 specific siRNA (siRab20) did not affect the phagocytosis of Escherichia coli (E. coli), but substantially impaired the intracellular killing of the ingested E. coli in BLP-tolerized macrophages. Furthermore, Rab20 was associated with GFP-E. coli containing phagosomes, and BLP tolerization resulted in the enhanced maturation of GFP-E. coli-containing phagosomes associated with Rab20 and strong lysosomal acidification. The knockdown of Rab20 substantially diminished lysosome acidification and disturbed the fusion of GFP-E. coli containing phagosomes with lysosomes in BLP-tolerized macrophages. These results demonstrate that Rab20 plays a critical role in BLP tolerization-induced augmentation of bactericidal activity via promoting phagosome maturation and the fusion of bacteria containing phagosomes with lysosomes.

Data-Independent Acquisition-Based Quantitative Proteomics Analysis Reveals Dynamic Network Profiles during the Macrophage Inflammatory Response.

Understanding of the molecular regulatory mechanisms underlying the inflammatory response is incomplete. The present study focuses on characterizing the proteome in a model of inflammation in macrophages treated with lipopolysaccharide (LPS). A total of 3597 proteins are identified in macrophages with the data-independent acquisition (DIA) method. Bioinformatic analyses reveal discrete modules and the underlying molecular mechanisms, as well as the signaling network that modulates the development of inflammation. It is found that a total of 87 differentially expressed proteins are shared by all stages of LPS-induced inflammation in macrophages and that 18 of these proteins participate in metabolic processes by forming a tight interaction network. Data support the hypothesis that ribosome proteins play a key role in regulating the macrophage response to LPS. Interestingly, conjoint analyses of the transcriptome and proteome in macrophages treated with LPS reveal that the genes upregulated at both the mRNA and protein levels are mainly involved in inflammation and the immune response, whereas the genes downregulated are significantly enriched in metabolism-related processes. These results not only provide a more comprehensive understanding of the molecular mechanisms of inflammation mediated by bacterial infection but also provide a dynamic proteomic resource for further studies.

The novel methyltransferase SETD4 regulates TLR agonist-induced expression of cytokines through methylation of lysine 4 at histone 3 in macrophages.

The production of inflammatory cytokines is closely related to pathogen-associated molecular pattern (PAMP)-triggered activation of the Toll-like receptor (TLR), intracellular signal transduction pathways such as MAPK and NF-κB, and histone modifications. Histone methylation, a type of histone modifications, is mainly accomplished by a class of SET family proteins containing highly conserved SET domains. In the present study, we found that SET domain-containing protein 4 (SETD4) regulated inflammatory cytokines in response to TLR agonists. LPS stimulation led to the enhanced SETD4 expression, while the increased IL-6 and TNF-α release from LPS-stimulated RAW264.7 cells was attenuated by depletion of SETD4 using RNA interference. The results were further confirmed in BMDMs and pMφ isolated from SETD4-deficient mice where SETD4-/- macrophages treated with LPS, BLP or Poly(I:C) showed down-regulated IL-6 and TNF-α mRNA and protein levels when compared with SETD4+/+ macrophages. Moreover, the mRNA levels of all NF-κB-dependent genes including IL-1ß, IL-10, NFKBA, DUSP1, CCL2, CCL5, and CXCL10 in SETD4-/- macrophages were substantially reduced. To further clarify the regulatory mechanism(s) by which SETD4 modulates inflammatory cytokines, we examined the effect of SETD4 on the activation of MAPK and NF-κB signalling pathways, and found that knockout of SETD4 had no effect on phosphorylation of p38, ERK, JNK, p65, and IκBα. Notably, SETD4 translocated quickly from the cytosol to the nucleus upon LPS stimulation, suggesting that SETD4 may exert its regulatory function downstream of the MAPK and NF-κB pathways. To characterize this, we performed an in vitro HMTase assay to measure histone methyltransferase (HMTase) activity of SETD4. H3K4me1 and H3K4me2 levels were enhanced dramatically with the supplementation of SETD4, whereas both H3K4me1 and H3K4me2 were strongly attenuated in SETD4-/- BMDMs. Moreover, the LPS-stimulated recruitment of H3K4me1 and H3K4me2 at both TNF-α and IL-6 promoters was severely impaired in SETD4-/- BMDMs. Collectively, these results demonstrate that SETD4 positively regulates IL-6 and TNF-α expression in TLR agonist-stimulated macrophages by directly activating H3K4 methylation.

The natural antisense transcript NATTD regulates the transcription of decapping scavenger (DcpS) enzyme.

Natural antisense transcripts (NATs) are transcribed from the opposite strand of other genes. Most of them are noncoding RNAs. They have been reported to play important roles in a variety of biological processes. In this study, we identified a novel NAT, NATTD, which is partially complementary to both the TIRAP/Mal and DcpS genes. Interestingly, NATTD only positively regulates the expression of DcpS, a decapping scavenger enzyme which is a promising therapeutic target for spinal muscular atrophy. But it has no obvious effects on the expression of TIRAP/Mal gene. The NATTD transcript primarily resides in the nucleus and does not alter the mRNA stability of DcpS. Instead, it is required for the recruitment of RNA polymerase II at the mouse DcpS promoter. Chromatin immunoprecipitation assays revealed that knocking-down NATTD transcript with shRNA enhanced the H3K27-Me3 modification at the DcpS promoter. In summary, our studies identified NATTD as a regulator of DcpS transcription through epigenetic mechanisms.

H3K4 Methylation Regulates LPS-Induced Proinflammatory Cytokine Expression and Release in Macrophages.

Histone methylation is an important epigenetic mechanism that plays an essential role in regulating gene expression in mammalian cells. To understand its influence on inflammation, methylation of H3K4, H3K9, H3K36, H3K79, and H4K20, the most common histones methylated in the inflammatory response was analyzed in murine RAW264.7 cells and bone marrow-derived macrophages (BMDMs) upon lipopolysaccharide (LPS) stimulation. LPS stimulation resulted in enhanced methylation at H3K4 and H3K9 in both RAW264.7 and BMDMs. To further confirm whether LPS-stimulated H3K4me2 and H3K9me2 were responsible for subsequent proinflammatory cytokine expression, the recruitment of H3K4me2 and H3K9me2 at the promoters of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) was assessed. H3K4me2, but not H3K9me2, was enriched at the promoters of both IL-6 and TNF-α. Furthermore, LPS-stimulated gene expression and release of IL-6 and TNF-α were markedly suppressed in macrophages by MTA, a specific inhibitor of H3K4 methylation. These results demonstrate that histone methylation, in particular H3K4me2, plays a critical role in the regulation of LPS-induced expression and release of IL-6 and TNF-α.

Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-ß to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively.

Activation of IKKß is the key step in canonical activation of NF-κB signaling. Extensive work has provided insight into the mechanisms underlying IKKß activation through the identification of context-specific regulators. However, the molecular processes responsible for its negative regulation are not completely understood. Here, we identified KLHL21, a member of the Kelch-like gene family, as a novel negative regulator of IKKß. The expression of KLHL21 was rapidly down-regulated in macrophages upon treatment with proinflammatory stimuli. Overexpression of KLHL21 inhibited the activation of IKKß and degradation of IκBα, whereas KLHL21 depletion via siRNA showed the opposite results. Coimmunoprecipitation assays revealed that KLHL21 specifically bound to the kinase domain of IKKß via its Kelch domains and that this interaction was gradually attenuated upon TNFα treatment. Furthermore, KLHL21 did not disrupt the interaction between IKKß and TAK1, TRAF2, or IκBα. Also, KLHL21 did not require its E3 ubiquitin ligase activity for IKKß inhibition. Our findings suggest that KLHL21 may exert its inhibitory function by binding to the kinase domain and sequestering the region from potential IKKß inducers. Taken together, our data clearly demonstrate that KLHL21 negatively regulates TNFα-activated NF-κB signaling via targeting IKKß, providing new insight into the mechanisms underlying NF-κB regulation in cells.

[The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody].

OBJECTIVE: To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein. METHODS: The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund's adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting. RESULTS: The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells. CONCLUSION: The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.

A differential role of macrophage TRPM2 channels in Ca²âº signaling and cell death in early responses to H2O2.

Reactive oxygen species such as H2O2 elevates the cytosolic Ca²âº concentration ([Ca²âº]c) and causes cell death via poly(ADPR) polymerase (PARP) activation, which also represents the primary mechanism by which H2O2 activate the transient receptor potential melastatin-related 2 (TRPM2) channel as a Ca²âº-permeable channel present in the plasma membrane or an intracellular Ca²âº-release channel. The present study aimed to define the contribution and mechanisms of the TRPM2 channels in macrophage cells in mediating Ca²âº signaling and cell death during initial response to H2O2, using mouse peritoneal macrophage, RAW264.7, and differentiated THP-1 cells. H2O2 evoked robust increases in the [Ca²âº]c, and such Ca²âº responses were significantly greater at body temperature than room temperature. H2O2-induced Ca²âº responses were strongly inhibited by pretreatment with PJ-34, a PARP inhibitor, and largely prevented by removal of extracellular Ca²âº. Furthermore, H2O2-induced increases in the [Ca²âº]c were completely abolished in macrophage cells isolated from trpm2-/- mice. H2O2 reduced macrophage cell viability in a duration- and concentration-dependent manner. H2O2-induced cell death was significantly attenuated by pretreatment with PJ-34 and TRPM2 channel deficiency but remained significant and persistent. Taken together, these results show that the TRPM2 channel in macrophage cells functions as a cell surface Ca²âº-permeable channel that mediates Ca²âº influx and constitutes the principal Ca²âº signaling mechanism but has a limited, albeit significant, role in cell death during early exposure to H2O2.

[Construction of pNTAP-MK2 eukaryotic expression plasmid and establishment of a cell line for its stable expression].

OBJECTIVE: To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2. METHODS: The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay. RESULTS: The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei. CONCLUSION: The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.

Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions.

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptor's sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.

Requirement for the N-terminal coiled-coil domain for expression and function, but not subunit interaction of, the ADPR-activated TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2) proteins form multiple-subunit complexes, most likely homotetramers, which operate as Ca2+-permeable, nonselective cation channels activated by intracellular ADP-ribose (ADPR) and oxidative stress. Each TRPM2 channel subunit is predicted to contain two coiled-coil (CC) domains, one in the N-terminus and the other in the C-terminus. Our recent study has shown that the C-terminal CC domain plays an important, but not exclusive, role in the TRPM2 channel assembly. This study aimed to examine the potential role of the N-terminal CC domain. Domain deletion dramatically reduced protein expression and abolished ADPR-evoked currents but did not alter the subunit interaction. Deletion of both CC domains strongly attenuated the subunit interaction, confirming that the C-terminal CC domain is critical in the subunit interaction. Glutamine substitutions into individual hydrophobic residues at positions a and d in the heptad repeats to disrupt the CC formation had no effect on protein expression, subunit interaction, or ADPR-evoked currents. Mutation of Ile(658) to glutamine, which did not perturb the CC formation, decreased ADPR-evoked currents without affecting protein expression, subunit interaction, or membrane trafficking. These results collectively suggest the requirement for the N-terminal CC domain for protein expression and function, but not subunit interaction, of the TRPM2 channel.

Inhibitory interaction between P2X4 and GABA(C) rho1 receptors.

Reciprocal functional inhibition between P2X and GABA(A/C) receptors represents a novel mechanism fine-tuning neuronal excitability. However, the participating receptors and underlying mechanisms are not fully understood. P2X(4) receptor is widely found in neurons that express GABA(C) rho1 receptor. Thus, we co-expressed P2X(4) and rho1 receptors in HEK293 cells and, using patch-clamp recording, examined whether they have mutual functional inhibition. Currents evoked by simultaneous application of ATP and GABA (I(ATP+GABA)) were significantly smaller compared to the addition of I(ATP) and I(GABA). Furthermore, I(ATP) were strongly suppressed during rho1 receptor activation. Similarly, I(GABA) were greatly attenuated during P2X(4) receptor activation. Such mutual inhibition was absent in cells only expressing P2X(4) or rho1 receptor. Taken together, these functional data support negative cross-talk between P2X(4) and rho1 receptors.

Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2.

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

[The molecular mechanism of survivin expression in activated human peripheral lymphocytes].

AIM: Investigate the molecular mechanism of regulating survivin expression and related signal transduction pathway, molecular cascade reaction and biological effects in activated PBMC. METHODS: The expression of survivin and related proteins were detected by Western blot in PBMC stimulated by PHA and rhIL-2 with or without JAK inhibitor-AG490 treatment, and FCM was performed to analyze cell cycle and cell division. RESULTS: Our results indicated that molecular and cellular reactions in PBMC activated by PHA and rhIL-2 were dependent on time series. At first, the phosphorylation of Stat3 and Stat5 were observed, then, protein levels of CyclinD3 and CyclinE increased, and the stimulated PBMC began to enter to S phage with survivin protein expression was initiated, which at last resulted in cell division with dramatically increasing expression of survivin protein. AG490 could significantly inhibit all these reactions but had no effect on the expressions of the cell cycle inhibitor-P21 and anti-apoptosis protein-Bcl-2. CONCLUSION: The expression of survivin in stimulated PBMC was dependent on the primarily activated JAK-STAT pathway, which upregulated CyclinD3 and CyclinE protein levels, initiated the cell cycle progression, and induced cell cycle-dependent survivin expression, and so survivin was involved in cell division and cell proliferation.

Intracellular coiled-coil domain engaged in subunit interaction and assembly of melastatin-related transient receptor potential channel 2.

TRPM2 channels, activated by adenosine diphosphoribose and related molecules, are assembled as oligomers and most likely tetramers. However, the molecular determinants driving the subunit interaction and assembly of the TRPM2 channels are not well defined. Here we examined, using site-directed mutagenesis in conjunction with co-immunoprecipitation and patch clamp recording, the role of a coiled-coil domain in the intracellular C terminus of TRPM2 subunit in subunit interaction and channel assembly. Deletion of the coiled-coil domain resulted in severe disruption of the subunit interaction and substantial loss of the adenosine diphosphoribose-evoked channel currents. Individual or combined mutations to glutamine of the hydrophobic residues at positions a and d of the abcdef heptad repeat, key residues for protein-protein interaction, significantly reduced the subunit interaction and channel currents; the mutational effects on the subunit interaction and channel currents were clearly correlated. Furthermore, deletion of the coiled-coil domain in a pore mutant subunit abolished its dominant negative phenotypic functional suppression. These results provide strong evidence that the coiled-coil domain is critically engaged in the TRPM2 subunit interaction and such interaction is required for assembly of functional TRPM2 channel. The coiled-coil domain, which is highly conserved within the TRPM subfamily, may serve as a general structural element governing the assembly of TRPM channels.

Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function.

TRPM2 proteins belong to the melastatin-related transient receptor potential or TRPM subfamily and form Ca(2+)-permeable cationic channels activated by intracellular adenosine diphosphoribose (ADPR). The TRPM2 channel subunit, like all its close relatives, is structurally homologous to the well-characterized voltage-gated potassium channel subunits, each containing six transmembrane segments and a putative pore loop between the fifth and sixth segments. Nevertheless, the structural elements determining the TRPM2 channel functions are still not well understood. In this study, we investigated the functional role of two conserved cysteine residues (at positions 996 and 1008) in the putative pore region of the human TRPM2 by site-directed mutagenesis, combined with electrophysiological and biochemical approaches. Expression of wild-type hTRPM2 channels in human embryonic kidney (HEK-293) cells resulted in robust ADPR-evoked currents. Substitution of cysteine with alanine or serine generated mutant channels that failed to be activated by ADPR. Furthermore, experiments done by Western blot analysis, immunocytochemistry, biotin labeling, and coimmunoprecipitation techniques showed no obvious changes in protein expression, trafficking or membrane localization, and the ability to interact with neighboring subunits that is required for channel assembly. Coexpression of wild-type and mutant subunits significantly reduced the ADPR-evoked currents; for the combination of wild-type and C996S mutant subunits, the reduction was approximately 95%, indicating that incorporation of one or more nonfunctional C996S subunits leads to the loss of channel function. These results taken together suggest that the cysteine residues in the pore region are obligatory for TRPM2 channel function.

[Inhibiting expression of human telomerase reverse transcriptase promotes degradation of survivin protein].

BACKGROUND & OBJECTIVE: The expression of Survivin in cancer cells highly correlates with that of human telomerase reverse transcriptase (hTERT). Both of them are ideal targets for cancer gene therapy. This study aimed to clarify if they regulate each other in cancer cells. METHODS: The expressions of Survivin and hTERT in HeLa S3 cells were inhibited by antisense oligonucleotide respectively. Activity of telomerase was detected by telomerase repeat amplification (TRAP) assay. Protein and mRNA levels of Survivin were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. Proliferation of HeLa S3 cells was analyzed by MTT assay. RESULTS: Inhibiting the expression of Survivin in HeLa S3 cells had no effects on telomerase activity. Inhibiting the expression of hTERT by antisense oligonucleotide No.14 decreased protein level of Survivin, which was negatively correlated with the concentration of No.14 (200-1 000 nmol/L), but didn't change mRNA level of survivin. The decrease of Survivin level was inhibited by proteasome inhibitor lactacystin and MG132. Furthermore, simultaneous inhibition of hTERT and survivin co-efficiently inhibited proliferation of HeLa S3 cells. CONCLUSION: Inhibiting the expression of hTERT in HeLa S3 cells promotes ubiquitin-proteasome degradation of Survivin.

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

[Mechanism of G2/M cell cycle arrest before apoptosis in leukemia cell line HL-60 induced by proteasome inhibitor MG132].

BACKGROUND & OBJECTIVE: Proteasome inhibitor is a kind of potential anti-tumor drug,it can induce apoptosis in various tumor cells. This study was designed to investigate the molecular mechanism of apoptosis and G(2)/M arrest in leukemia cell line HL-60 induced by proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO). METHODS: Apoptosis in HL-60 cells was observed under fluorescent microscope, flow cytometry and immunoblot were used to analyze cell apoptosis, cell cycle arrest, and the mechanisms. RESULTS: MG132 (2 micromol/L)induced apoptosis in HL-60 cells after 24-h treatment. Meanwhile, HL-60 cells were arrested at G(2)/M phase before apoptosis after induced by MG132. The percentage of G(2)/M phase in MG132-treated HL-60 cells at 12 h was 63.42+/-2.02,while that in untreated cells was 7.29+/-3.01 (P< 0.01). The percentage of apoptosis in MG132-treated HL-60 cells at 24 h was 16.67+/-1.48, while untreated cells had no death (P< 0.01). Compared to the treatment with MG132 only, caffeine (2 mmol/L) exposure can reduce G(2)/M arrest and apoptosis in MG132-treated HL-60 cells. Expression of cyclin-dependent kinase inhibitor p21waf/cip1 up-regulated after treated with MG132 for 3 h, but no p53 or p27 detected. CONCLUSIONS: Proteasome inhibitor MG132 can induce G2/M arrest before the apoptosis appeared in HL-60 cells. The obvious up-regulation of p21 indicated that it is p21(waf/cip1), but not p53 or p53-related proteins,that involved in the regulation of G(2)/M arrest and subsequent apoptosis induced by MG132 in HL-60 cells.

Effects to HeLa Cells of the Inhibition of Survivin by Antisense RNA.

To study the function of survivin in cancer cells, survivin cDNA was amplified from HeLa cells by RT-PCR. Then the coding fragment was subcloned into an inducible eukaryotic expression plasmid pHC in reverse direction.The obtained plasmid pHSC was transfected into HeLa cells by Lipofectamine. An inducible cell line which can express antisense RNA of survivin with 2 mmol/L Zn(2 ) was obtained by the selection with 800 nmol/L G418 solution. The expression of antisense RNA of survivin inhibited the expression of survivin protein. Inhibition of survivin expression suppressed the proliferation of HeLa cells and its cell cycle. And it also sensitized HeLa cells to chemotherapeutics.