[Effects of Quercetin on Chronic Myeloid Leukemia Cell Line Resistant to Imatinib and Its Mechanism].

OBJECTIVE: To explore the growth inhibitory effect of quercetin on imatinib-resistant chronic myeloid leukemia cell lines and to clarify its involved mechanisms. METHODS: The cell viability was detected by trypan blue Staining, percentage of apoptotic cells and cell cycle distribution were detected by flow cytometry, the protein expression was detected by Western blot. RESULTS: Both inhibitory effect of proliferation and apoptosis-inducing effect were similar between the imatinib-resistant and -sensitive cell lines treated with 25 µmol/L quercetin for 24 hours and with arrest of cell cycle at G2/M phase. Quercetin could not change the expression of BCR-ABL. The expression of γ-H2AX was markedly enhanced and the phosphorylation of JNK up-regulated by quercetin in both imatinib-resistant and imatinib-sensitive cell lines. CONCLUSION: The growth of imatinib-resistant cells can be inhibited by quercetin, and the apoptosis of cells can be induced by quercetin, which may be related to cell cycle arrest in G2/M. The DNA damage and up-regulation of p-JNK may be involved in these processes.

RIG-G inhibits the proliferation of NB4 cells and propels ATRA-induced differentiation of APL cells.

RIG-G (retinoic acid-induced gene G) was originally identified in ATRA (all-trans retinoic acid)-treated NB4 acute promyelocytic leukemia (APL) cells. It was induced to expression by ATRA along with the differentiation of the cells. However, little is known about its role(s). Here, we established a RIG-G stably expression transformant of NB4 cells. By using the transformant, we showed that expression of RIG-G in NB4 cells not only arrested the cells at G1/G0 transition phase and inhibited their proliferation, but also markedly drive the maturation of NB4 cells in the presence of very low concentration of ATRA (10(-9)mol/L). What's more, by detecting the expression of RIG-G in fresh primary bone marrow mononuclear cells of APL patients in different morbid states, we found high RIG-G expression level in complete remission patients, while low level in untreated or relapsed patients. These results indicated that RIG-G level was high in maturated cells and low in blast cells, and suggested that RIG-G might play a role in the differentiation of bone marrow hemocytes in vivo.

Rig-G negatively regulates SCF-E3 ligase activities by disrupting the assembly of COP9 signalosome complex.

We previously showed that Rig-G, an antiproliferative protein induced by interferon, can sequester CSN5 protein in the cytoplasm. Here, we report that Rig-G can destroy the functions of CSN5-containing COP9 signalosome (CSN), a highly conserved multiprotein complex implicated in protein deneddylation, deubiquitination, and phosphorylation. By damaging integrity and stability of the CSN complex, Rig-G can dramatically reduce the cellular content of CSN complex and inhibit its regulatory roles in assembly and activation of cullin-RING ubiquitin E3 ligases (CRL). Furthermore, Rig-G can cause excessive activation of CRL through inhibition of CSN-mediated deneddylation, largely decreasing protein levels of Cul1 and ßTrCP, two important subunits of SCF (Skp1-Cul1-F-box protein)-E3 ligase. Rig-G can also attenuate the ability of CSN to recruit USP15 and impair CSN-associated deubiquitination. Increased autoubiquitination of ßTrCP and concomitant accumulation of target substrates (such as IκBα) are observed in Rig-G-expressing cells. Taken together, our findings reveal for the first time the negative regulation of Rig-G on SCF-E3 ligase activities through disrupting CSN complex, not only contributing to further investigation on biological functions of Rig-G, but also leading to better understanding of the CSN complex as a potential target in tumor diagnosis and treatment.

[Mechanism of apoptosis synergistically induced by bortezomib combined with cytarabine in U937 cell line].

This study was aimed to further explore the apoptosis-inducing effect of bortezomib combined with cytarabine (Ara-C) on U937 cell line. Proliferation and apoptosis in U937 cells treated with bortezomib and/or Ara-C were assessed by cell count. Cell cycle distribution and reactive oxygen species (ROS) production level were measured by using flow cytometry. Cell signaling pathway related to apoptosis was analyzed by Western blot. The results showed that 10 nmol/L bortezomib combined with 50 nmol/L Ara-C significantly inhibited U937 cell proliferation. These two drug combination synergistically induced apoptosis in U937 cells, significantly increased cellular ROS level, and up-regulated the expression of phosphorylated form of JNK and P38 and down-regulated phosphorylation of ERK. It is concluded that the apoptosis of U937 cells synergistically induced by bortezomib combined with low concentration Ara-C is possibly associated with up-regulation of phosphorylated form of JNK, P38 and down-regulation of phosphorylation of ERK induced by increase of ROS, resulting in decrease of mitochondrial potential.

[Effect of bortezomib and low concentration cytarabine on apoptosis in U937 cell line].

This study was aimed to explore the effect of bortezomib and low concentration cytarabine (Ara-C) on proliferation and apoptosis in U937 cell line and its mechanism. The proliferation and apoptosis of U937 cells treated with bortezomib (10 nmol/L) and(or) Ara-C (50 nmol/L) were observed by cell count, cell morphology, flow cytometry and Western blot. The results showed that bortezomib and Ara-C alone inhibited U937 cell proliferation. The inhibitory effect was enhanced by combination of these two drugs, the inhibitory rates of U937 cell proliferation were (55.00 ± 2.81)% and (70.02 ± 3.33)% after treatment for 24 h and 48 h, respectively. Bortezomib and Ara-C synergistically induced apoptosis and decreased mitochondrial membrane potential in U937 cells. The percentage of Rhodamin123 positive cells was (38.70 ± 1.54)%. Bortezomib and Ara-C also synergistically induced activation of caspase-9, caspase-8 and caspase-3. It is concluded that the bortezomib and low concentration Ara-C synergistically induced apoptosis in U937 cells, mainly through mitochondrial pathway, and possibly through death receptor pathway.

[Role of auto-secreted interferon α in all-trans retinoic acid-induced expression of RIG-G gene].

OBJECTIVE: To explore the relationship between interferon (IFN) α and all-trans retinoic acid (ATRA)-induced signaling pathways in the expression of retinoic acid-induced gene G (RIG-G). METHODS: Acute promyelocytic leukemia cell line NB4 and signal transducer and activator of transcription (STAT)1-deficient U3A cells were used. The protein levels of tyrosine-phosphorylated STAT2 in ATRA-treated NB4 cells were detected by Western blot. The culture supernatants of NB4 cells treated with ATRA for different time or U3A cells transfected with interferon regulatory factor (IRF)-1 were respectively collected. And the concentration of IFN-α was determined by enzyme-linked immunosorbent assay (ELISA). The effects of NB4 cell culture supernatants on the phosphorylation of STAT2 and the expression of RIG-G were detected by Western blot. RESULTS: The level of phosphorylated-STAT2 was obviously up-regulated in NB4 cells treated with ATRA for 72 hours, as well as the concentration of IFN-α in culture supernatants. The concentration of IFN-α increased from (1.5 ± 0.5) pg/ml in the untreated group to (7.6 ± 0.3) pg/ml (P < 0.05). After a 96-hour treatment, the concentration of IFN-α was up to (63.8 ± 5.8) pg/ml. And these culture supernatants could induce the tyrosine phosphorylation of STAT2 and up-regulate the protein level of RIG-G. As for U3A cells transfected with IRF-1, the concentration of IFN-α from the culture supernatant also increased 3-fold versus the control group transfected with empty vectors [(8.8 ± 1.4) pg/ml vs (3.4 ± 0.4) pg/ml, P < 0.05]. CONCLUSIONS: RIG-G gene expression is closely correlated with the cross-talk between ATRA and IFN-α-induced signaling pathways. ATRA increases the secretion of IFN-α by up-regulating the protein level of IRF-1. Then the secreted IFN-α may induce the phosphorylation of STAT2 and reinforce the expression of RIG-G.

Intact JAK-STAT signaling pathway is a prerequisite for STAT1 to reinforce the expression of RIG-G gene.

We previously reported that IRF-9/STAT2 functional interaction could drive the expression of retinoic acid-induced gene G (RIG-G), independently of STAT1 and the classical JAK-STAT pathway, providing a novel alternative pathway for interferons (IFN) to mediate their multiple biological properties. In addition, we also found that IRF-1 could regulate RIG-G induction as well as the expression of IRF-9 and STAT2 in some cases. But the mechanisms by which IRF-1 exerted its action remained to be elucidated. Here, we showed that STAT1 could significantly enhance the effects of the IRF-9/STAT2 complex or IRF-1 on RIG-G induction through an activated JAK-STAT pathway, though it was not essential for RIG-G expression. In STAT1-deficient U3A cells, IRF-1 could induce RIG-G expression via the IFN-stimulated response elements in the RIG-G gene promoter, but it failed to upregulate IRF-9 and STAT2 unless the U3A cells were reconstituted by exogenous STAT1. In STAT1-expressing cells, IRF-1 indirectly activated RIG-G expression through an IRF-9/STAT2-dependent manner. Taken together, we concluded that the expression of RIG-G was independent on the classical JAK-STAT pathway, but could be greatly increased by it. This work will be of great benefit to us for a better understanding of the mechanisms on RIG-G gene expression regulation.

[Expression of ifi56 gene in ATRA-induced APL cell differentiation and construction of ifi56 gene eukaryotic expression plasmid].

This study was purposed to investigate the expression of ifi56 gene in the ATRA-induced acute promyelocytic leukemia (APL) NB4 cell differentiation and to construct the eukaryotic expression plasmid of ifi56 gene. RT-PCR was used to detect the expression of ifi56 in NB4 cells treated with ATRA for different time. Human ifi56 cDNA was amplified by RT-PCR and cloned into pEGFP-C1 vector, then was transfected into 293T cells. The expression of the recombinant protein in 293T cells was detected by Western blot. The localization of IFI56 protein was observed by fluorescence microscopy. The results showed that the ifi56 mRNA was almost undetectable in untreated NB4 cells, but it significantly increased after ATRA treatment for 72 hours. The cDNA fragment of ifi56 was inserted into the expressing plasmid pEGFP-C1 successfully. The expression of EGFP-IFI56 fusion protein with a molecular weight about 83 kD was detected by Western blot. The EGFP-IFI56 protein was localized in cytoplasm mainly. It is concluded that the expression of ifi56 is enhanced significantly when the differentiation of APL cells was induced by ATRA. Gene ifi56 is successfully cloned into eukaryotic expression vector and the fusion protein is expressed in the cytoplasm mainly.

[Role of the interferon-stimulated response elements I/II in expression regulation of the retinoic acid induced gene G].

OBJECTIVE: To study the regulatory role of interferon-stimulated response elements (ISREs) located on the retinoic acid-induced gene G (RIG-G) promoter in RIG-G expression. METHODS: By using point mutation technique, the authors constructed the wide type and site mutant reporter gene plasmids according to the ISRE sequence on RIG-G promoter, and detected the functional activities by luciferase reporter assay. RESULTS: Mutation in ISRE II alone had no obvious effect on the expression of the reporter gene, whereas mutation in ISRE I dramatically inhibited the transactivity of RIG-G promoter. Mutation in both ISRE I and ISRE II resulted in complete loss of its response to the transcription factors for the reporter gene. CONCLUSION: Both ISRE I and ISRE II on the RIG-G promoter are the binding sites for the complex of transcription factors. They are required for RIG-G expression, and ISRE I has a preferential role over ISRE II.

[A novel molecular mechanism of interferon alpha-regulated expression of retinoic acid-induced gene G].

OBJECTIVE: To investigate the molecular mechanisms by which IFN-alpha regulated retinoic acid-induced gene G (RIG-G) expression. METHODS: The expression of STAT1, p-STAT1 and RIG-G in IFN-alpha-treated NB4 cells was detected by Western blot. The roles of STAT1, STAT2 and IRF-9 in IFN-alpha-induced RIG-G expression were analyzed in STAT1-null U3A cells by cell transfection, reporter gene assay, co-immunoprecipitation and chromatin immunoprecipitaion. RESULTS: In U3A cells, only when STAT2 and IRF-9 were co-transfected, the luciferase activities of RIG-G promoter-containing reporter gene could be highly increased about 8-fold compared with that in the control group. Moreover, in the absence of IFN-alpha, similar effects were observed in either IRF-9 co-transfected with wild type or mutant form of STAT2, whereas IFN-alpha could increase the transactivation activity of wild type STAT2 and IRF-9 by 6-fold compared with that without IFN-alpha, but had no effect on mutant STAT2. In addition, STAT2 could interact with IRF-9 and bind to the RIG-G promoter. CONCLUSION: STAT2 may interact with IRF-9 in a STAT1-independent manner. The complex STAT2/IRF-9 is the key factor mediating the expression of RIG-G gene regulated by IFN-alpha. This is a novel signal transduction cascade for IFN which is different from the classical JAK-STAT pathway.

[Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid].

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

IRF-9/STAT2 [corrected] functional interaction drives retinoic acid-induced gene G expression independently of STAT1.

Retinoic acid-induced gene G (RIG-G), a gene originally identified in all-trans retinoic acid-treated NB4 acute promyelocytic leukemia cells, is also induced by IFNalpha in various hematopoietic and solid tumor cells. Our previous work showed that RIG-G possessed a potent antiproliferative activity. However, the mechanism for the transcriptional regulation of RIG-G gene remains unknown. Here, we report that signal transducer and activator of transcription (STAT) 2 together with IFN regulatory factor (IRF)-9 can effectively drive the transcription of RIG-G gene by their functional interaction through a STAT1-independent manner, even without the tyrosine phosphorylation of STAT2. The complex IRF-9/STAT2 is both necessary and sufficient for RIG-G gene expression. In addition, IRF-1 is also able to induce RIG-G gene expression through an IRF-9/STAT2-dependent or IRF-9/STAT2-independent mechanism. Moreover, the induction of RIG-G by retinoic acid in NB4 cells resulted, to some extent, from an IFNalpha autocrine pathway, a finding that suggests a novel mechanism for the signal cross-talk between IFNalpha and retinoic acid. Taken together, our results provide for the first time the evidence of the biological significance of IRF-9/STAT2 complex, and furnish an alternative pathway modulating the expression of IFN-stimulated genes, contributing to the diversity of IFN signaling to mediate their multiple biological properties in normal and tumor cells.

[Effects of PML-RARalpha on cAMP-induced AML cell differentiation].

To explore the molecular mechanisms of acute promyelocytic leukemia cell differentiation induced by cAMP combined with low-dose As2O3, the PR9 cell line, which was stably transfected by PML-RARa fusion gene, was used as in vitro model. The effects of PML-RARa on cAMP-induced AML cell differentiation were evaluated according to cell growth, cell morphology, cell surface antigen as well as luciferase reporter gene assay, in the cells before and after the treatment with cAMP and/or As2O3. The results showed that cAMP alone could slightly increase the expression of CD11b in the PR9 cells expressing the PML-RARa fusion protein, but could not induce these cells to differentiate. The cells presented the terminal differentiation morphology and significantly increased CD11b expression only under the treatment of cAMP combined with As2O3. In addition, PML-RARa had strong inhibitory activity on the transcription of the reporter gene containing cAMP response elements. In conclusions, the PML-RARa fusion protein could dramatically block the signaling pathway of cAMP during the AML cell differentiation.

[Molecular mechanism of the inhibitory effect of retinoic acid-induced gene G protein on tumor cell proliferation].

OBJECTIVE: To investigate the molecular mechanisms of anti-proliferative effect of retinoic acid-induced gene G (RIG-G) protein on tumor cells. METHODS: HA-RIG-G expression plasmid and FLAG-Jun activating binding protein 1 (JAB1) expression plasmid were construction and transfected into the African green monkey kidney cells of the line CDS-7 and mouse fibroblast cells of the line NIH3T3. Western blotting was used to detect the p27 expression in the cells. Analysis, and Immunofluorescence staining was used to examine the distribution of JAB1 protein. Coimmunoprecipitation was used to analyze the interference of RIG-G on the function of JAB1 protein. RESULTS: Coimmunoprecipitation showed that when HA-RIG-G and FLAG-JAB1 were co-expressed, the RIG-G protein and JAB1 protein could be co-precipitated by the antibodies of the other side. RIG-G was able to interact with JAB1 and alter its intracellular localization and distribution. When JAB1 was transfected alone into the NIH3T3 cells, it dispersed in both nucleus and cytoplasm; however, when RIG-G and JAB1 were cotransfected, the nuclear JAB1 was markedly diminished and exhibited a partly co-localization with RIG-G in the cytoplasm. Western blotting showed that along with the increase of the dose of transfected JAB1 the amount of p27 in the cell; and along with the increase of co-transfected RIG-G gene expression plasmid the amount of p27 in the cells re-increased. CONCLUSION: RIG-G interacts with JAB1, thus resulting in JAB1 sequestration in the cytoplasm, disturbing the JAB1 normal function, interfering the JAB1-mediated p27 degradation, maintaining p27 protein stability so as to prevent cells from entering the cycle and inhibiting cell proliferation.

[cAMP analogue 8-CPT-cAMP inducing differentiation in the M2b subtype of acute myeloid leukemia cell line Kasumi-1].

This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.

[Two-photon excitation fluorescence of 5-ALA induced PpIX in DHL cells].

Two-photon fluorescence microscopy is a novel imaging technique, which is primarily sensitive to a specimen's response coming from an in-focus plane, thus has low photo-bleaching and photo-damage to biological samples. 5-ALA induced production of PpIX in DHL cells was excited by 820 nm femtosecond laser; two-photon excitation fluorescence of single cell was obtained in Lambda mode of laser scanning confocal microscope. The specific fluorescence intensity of PpIX which accumulated in DHL cells was measured at 2, 4 and 10 mmol x L(-1) concentration of 5-ALA with different incubation time, which reflected the kinetics of 5-ALA accumulated in DHL cells. Accumulation of PpIX in DHL cells was a dynamic change process. Biphasic alterations of PpIX accumulation were noted: PpIX content enhanced with the increasing time and reached the maximal value around 3 h, however PpIX content decreased in the subsequent incubation time. Results indicate that two-photon fluorescence based on laser scanning microscope can be a useful technology for studying the kinetics of 5-ALA induced PpIX production in DHL cells and other leukemia cells.

[In vitro study of the effects of CDA-II combined with cAMP on apoptosis induction in retinoic acid resistant acute promyelocytic leukemia cells].

OBJECTIVE: To investigate the effects of CDA-II alone or combined with cAMP on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells. METHODS: The RA-resistant cell line NB4-R2 was used as an in vitro model and treated with CDA-II alone or in combination with cAMP. Cell apoptosis was assessed by morphology observation, distribution of cellular DNA contents and sub-G1 cell population. The level of Bcl-2 was detected by flow cytometry, DNA "ladder" was detected by agarose-electrophoresis. RESULTS: CDA-II could induce NB4-R2 cell apoptosis through decreasing the level of cellular anti-apoptotic protein Bcl-2. cAMP could significantly enhance the role of CDA-II. Bcl-2 positive cell rates decreased to (15.1 +/- 4.8)% and (7.3 +/- 2.9)% in NB4-R2 cells treated with 1 mg/ml CDA-II plus 100 micromol/L cAMP for 48 h and 72 h, respectively. While 100 micromol/L of cAMP could decrease Bcl-2 positive NB4-R2 cells from (92.0 +/- 0.6)% to (75.3 +/- 2.0)%. CONCLUSIONS: CDA-II combined with cAMP could exert potent apoptotic effect on RA-resistant APL cells.

[Apoptosis of multiple myeloma cells induced by 8-Cl-cAMP in vitro].

The study was aimed to investigate the possible effects of 8-chloroadenosine 3', 5'-monophosphate (8-Cl-cAMP) on the multiple myeloma cells. The multiple myeloma cell line RPMI8226 was used as in vitro models. The effect on growth inhibition of RPMI8226 cells was evaluated by cell growth and viability curve. DNA fragment was visualized by agarose gel electrophoresis. The amount of apoptosis cells was measured by flow cytometry. Meanwhile Western blot assay were used to detect the change of several key cell cycle regulatory proteins CDK2 and cyclin E in these cells before and after the treatment. The results showed that low dose 8-Cl-cAMP (1-30 micromol/L) inhibited the proliferation and viability of RPMI8226 cells significantly. Agarose gel electrophoresis of DNA revealed the apoptosis characteristic "ladder" pattern. Apoptosis was also confirmed by flow cytometry. In addition, 8-Cl-cAMP was able to inhibit the cell growth through modulating expression of cell cycle regulators CDK2 and cyclin E. It is concluded that 8-cl-cAMP inhibits the proliferation and induce apoptosis of multiple myeloma cells effectively.

[Study on mechanisms of the expression regulation of interferon-induced gene RIG-G].

OBJECTIVE: To investigate the molecular mechanisms of the expression regulation of retinoic acidinduced gene G (RIG-G) by interferon alpha (IFNalpha). METHODS: RIG-G promoter region was analyzed by bioinformatics. The functional activities of RIG-G promoter with or without IFNalpha were detected by luciferase reporter assay and electrophoretic mobility shift assay (EMSA). RESULTS: RIG-G promoter region contained two well-conserved IFN-stimulated response elements (ISREs). Both ISRE I and ISRE II showed their effective binding abilities with signal transducer and activator of transcription 1 (STAT1). In HT1080 cells, in contrast with the empty plasmid pXP2, pXP2-A reporter construct containing intact ISRE I and ISRE II showed a significant higher baseline expression (1741.2 +/- 517.5) which could be further enhanced up to three-four folds by IFNalpha (5338.7 +/- 1226.9, P < 0.05). However, the luciferase activity of pXP2-A as well as its IFNalpha inducibility could be abrogated in STAT1-deficient U3A cells (from 1741.2 +/- 517.5 to 406.1 +/- 103.2, P < 0.05), indicating that the STAT1 protein was a prerequisite for the activities of ISRE I and ISRE II. CONCLUSION: ISREs present in RIG-G promoter region are molecular basis of IFNalpha induced RIG-G expression. RIG-G is a target gene directly regulated by STAT1 protein and should play a key role in IFNalpha signaling pathways.

[Utilization of the stable ectopic expression cell line as a model for the investigation of RIG-G gene].

OBJECTIVE: To investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein. METHODS: Ectopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins. RESULTS: RIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells. CONCLUSIONS: RIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.