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PubMed2003

Reduced hepatic tumor incidence in cyclin G1-deficient mice.

Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53.

Alkylating Agents↗
PubMed2003

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition.

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.

Antibodies, Monoclonal↗
PubMed1997

Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts.

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant, p21(Ha-Ras[Asn17]), is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminated the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest was also observed in cells in which Ras(Asn17) was induced after growth stimulation with a phorbol ester or alpha-thrombin and was mimicked by the addition late in the G1 phase of inhibitors for phosphatidylinositol-3-kinase. Ras-mediated downregulation of p27kip1 involved both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression did not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. We conclude that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase.

3T3 Cells↗
PubMed2000

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.

Animals↗
PubMed1996

Cytogenetic damage and the radiation-induced G1-phase checkpoint.

It is proposed that genomic integrity is preserved after DNA damage in a variety of ways. X irradiation induces a p53-dependent G1-phase cell cycle checkpoint which putatively allows time for repair of DNA damage. The p53 protein is also involved in the initiation of apoptosis after radiation-induced DNA damage, presumably leading to the elimination of lethally damaged cells from the irradiated population. To test the hypothesis that repair occurs in the additional time provided by the activation of the G1-phase checkpoint, we investigated whether the presence of a G1-phase arrest modified the frequency and type of chromosomal rearrangements at the first mitosis after irradiation. Isogenic cell lines derived from the same human glioma cell line, but differing in p53 status, were used. Purified G1-phase cells, isolated by centrifugal elutriation and X-irradiated, were studied. The wild-type p53 cell line demonstrated a dose-dependent arrest during G1 phase, as determined by flow cytometry. These cells remained in G1-phase as long as 48 h after irradiation. Cells expressing a dominant-negative p53 mutation accumulated to a much lesser extent in G1 phase after irradiation. Cells lacking the G1-phase checkpoint showed increased survival at all radiation doses. There were no significant differences in the type or frequency of total chromosomal aberrations in mitotic cells from either cell line after 1,2,4 or 6 Gy X rays, as measured by conventional cytogenetic analysis. There was an increase, however, in the number of reciprocal translocations in mitotic cells with mutant p53 (lacking a G1-phase checkpoint), as measured by fluorescence in situ hybridization with a chromosome 4-specific DNA library, but only after 6 Gy. The results suggest that the presence of a well-defined p53-dependent G1-phase arrest does not reduce chromosomal aberrations caused by low doses of ionizing radiation markedly, but may reduce the overall degree of survival by triggering other G1-phase events.

Apoptosis↗
PubMedDate not supplied

Co-effect of HLA-G1 and glycosyltransferases in reducing NK cell-mediated pig endothelial cell lysis.

Natural killer (NK) cells play an important role in xenograft rejection. The aim of this study was to evaluate the co-effect of human leukocyte antigen (HLA)-G1 expression and the remodeling of glycoantigens such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes related to NK cell-mediated direct cytotoxicity. Human peripheral blood mononuclear cells or an NK-like cell line, YT cells, was used as an effector and pig endothelial cells (PEC) as the target. A PEC transfectant with HLA-G1 was first prepared by the transfection of HLA-G1 and human beta2 microglobulin. Several new transfectants were then established by the transfection of glycosyltransferase to the HLA-G1 transfectant. The effect of HLA-G1 on NK cell-mediated PEC lysis was lower than that by the glycosyltransferases. Therefore, in the case of the co-transfectants except for HLA-G1+alpha2,6sialyltransferase, such as HLA-G1+N-acetylglucosaminyltransferase-III and HLA-G1+alpha1,2fucosyltransferase, the effect of HLA-G1 expression on NK-mediated killing appeared to be accounted for by the transfected glycosyltransferase activities and the reduced alpha-Gal expression on the cell surface. However, these transfectants showed significant reductions in direct NK cell-mediated cytotoxicity, compared with the single HLA-G1 transfectant. The results herein suggest that a combination of HLA-G1 and glycosyltransferases has considerable potential for the downregulation of NK cell-mediated cytolysis.

Animals↗
PubMed2003

Down-regulation of HLA-G1 cell surface expression in human cytomegalovirus infected cells.

PROBLEM: Down-modulation of human leukocyte antigen (HLA)-G1 cell surface expression by human cytomegalovirus (HCMV) has only been studied in cellular models expressing independent unique short (US) recombinant proteins, but not in the context of viral infection. To explore the level of HLA-G1 cell surface expression after HCMV infection and to investigate the influence of US viral proteins, we infected HLA-G1 expressing cells by HCMV laboratory strains. METHOD OF STUDY: Human U373-MG astrocytoma cells were transfected with HLA-G1 cDNA. Following HCMV infection, HLA-G1 cell surface expression of these transfectants was evaluated by flow cytometry and confocal microscopy, using an HLA-G specific monoclonal antibody, and compared with that of uninfected cells. US-deleted viruses were then used to evaluate the implication of US proteins. RESULTS: Using flow cytometry, it was found that HCMV infection of U373-G1 cells decreased HLA-G1 cell surface expression. Similar results were obtained with two different HCMV strains, namely Towne and AD169. Two color confocal microscopy staining further confirmed such HLA-G down-modulation in HCMV-infected cells stained for immediate early (IE1/2) nuclear proteins expression. Infection of U373-G1 cells with US-deleted HCMV strain had no effect on the level of cell surface HLA-G1 expression, thus demonstrating the US dependency of the HCMV-mediated down-regulation of HLA-G1. CONCLUSION: HCMV infection down-modulates HLA-G1 expression at the cell surface. This is likely to have functional consequences in case of HCMV uterine infection during pregnancy.

Cell Line↗
PubMed1997

Fission yeast pheromone blocks S-phase by inhibiting the G1 cyclin B-p34cdc2 kinase.

Yeast pheromones block cell cycle progression in G1 in order to prepare mating partners for conjugation. We have investigated the mechanism underlying pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe. We find that the G1-specific transcription factor p65cdc10-p72res1/sct1 which controls the expression of S-phase genes is fully activated in pheromone, unlike the analogous control in budding yeast. In contrast, the G1 function of p34cdc2 acting after activation of the G1-specific transcription is blocked. Pheromone inhibits the p34cdc2 kinase associated with both the G1-specific B-type cyclin p45cig2 and the B-type cyclin p56cdc13 and overexpression of p45cig2 or p47cdc13delta90 overcomes the pheromone-induced G1 arrest. G1 arrest is compromised in enlarged cells. We suggest that onset of S-phase is controlled by pheromone inhibiting the B-cyclin-associated kinase in G1, and that increasing cell size contributes to the mechanism for pheromone adaptation. Thus, pheromone in fission and budding yeast acts similarly in inhibiting the G1 cyclin-dependent kinase (CDK), but differs in its effects on the G1/S transcriptional control, suggesting that inhibition of CDKs may be a more general mechanism for the control of G1 progression compared with G1/S transcriptional control.

Blotting, Northern↗
PubMedDate not supplied

Multicenter evaluation of tendency-oriented perimetry (TOP) using the G1 grid.

PURPOSE: The G1-TOP program is a short automated perimetric strategy which sub-divides the G1 grid of 59 points into four sub-grids. Each point is tested only once, but each patient's response is used to modify that particular point and the surrounding ones from the remaining sub-grids. This study compared the results of the G1-TOP program with the Standard Bracketing strategy. METHODS: Eleven participating institutions provided data from 213 patients (406 eyes). The main group consisted of 284 glaucomas and 55 glaucoma suspects. Other groups included 31 eyes with neurological disorders, 20 with chorioretinal lesions and 16 normal eyes. Mean age was 62.7 +/- 15.4 (range 14-88) years. All subjects had previous perimetric experience and visual acuity better than 0.5. Examination included G1-Standard Bracketing and G1-TOP testing, in interchangeable order, with the Octopus 1-2-3 perimeter. RESULTS: The correlation coefficient for mean defect (MD) was 0.95. Standard error (YX) for MD, square root of loss variance (LV) and individual thresholds were 1.86 dB, 1.29 dB, and 4.72 dB, respectively. Mean sensitivity values were similar (difference 0.04 +/- 1.87 dB) (p>0.05). Mean duration for G1-TOP was 2.19 +/- 0.26 min, while G1-Standard Bracketing took 11.51 +/- 1.52 min (ratio 1/5.1, or a net reduction of 80.4%). The sensitivity of G1-TOP versus G1-Standard Bracketing was: glaucoma 77.1/78.5, glaucoma suspects 38.2/47.3, neurological disorders 87.1/87.1 and chorioretinal lesions 80.0/85.0. CONCLUSIONS: The G1-TOP program gave very similar results to G1-Standard Bracketing in only 20% of the time required by the standard strategy.

Adolescent↗
PubMed1994

Accelerated G1-transit following transient inhibition of DNA replication is dependent on two processes.

Accelerated G1 transit in HeLa cells develops after a temporary inhibition of DNA synthesis in the previous generation. G1 acceleration has been found to be dependent on (1) the cell-cycle position at the time of the inhibition and (2) the duration of the inhibition. The degree of G1 acceleration correlates with the position in S-phase at which DNA synthesis is blocked. G1 transit time decreases gradually beginning with early S blocks and reaches a minimum when DNA synthesis is inhibited at mid S-phase. A minimum of 4 h continuous inhibition at this position resulted in maximum G1 acceleration. Unbalanced growth, i.e., the nonspecific increase in total protein or RNA levels that results from inhibition of DNA synthesis, did not correlate with the decrease in G1 transit time. On the other hand, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed specific changes in four nuclear proteins of molecular weights 120, 66, 57, and 51 kDa in cells immediately following release from inhibition of DNA synthesis at mid S-phase. A Western blot analysis of cyclins D and E showed an increased accumulation of cyclin E at 0, 1, 5, 7, and 12 h following cell release from the mid S-phase block. Therefore, it is unlikely that changes in the ratio of protein/DNA or RNA/DNA are responsible for the decrease in G1 transit times as suggested by the unbalanced growth model of G1 acceleration. Instead, G1 acceleration may result from the accumulation during S-phase blockage, and subsequent retention through G2 and mitosis, of specific proteins required for cell transit through G1 in the next cell cycle.

Aphidicolin↗
PubMed1999

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells.

Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21(Cip1/Waf1) induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin-induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0-G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0-G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21(Cip1/Waf1), and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin-induced G2 arrest was relatively tight and correlated with the Thr-14/Tyr-15 phosphorylation of cyclin B-Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.

3T3 Cells↗
PubMed2002

Cyclin G1 associates with MDM2 and regulates accumulation and degradation of p53 protein.

BACKGROUND: Cyclin G1 is a transcriptional target of p53 and is induced by DNA damage in a p53 dependent manner. Analysis of cyclin G1 disrupted mice demonstrated that cyclin G1 is involved in many of the functions regulated by p53 such as apoptosis, growth control and check point regulation in response to DNA damage. The results suggest that the main role of cyclin G1 is to mediate or regulate the function of p53. RESULTS: Western blot analysis revealed that the accumulation of p53 protein during the initial 24 h period following DNA damage is reduced in cyclin G1-/- cells compared to wild-type cells. This decrease in p53 accumulation could be recovered by introducing a cDNA expressing cyclin G1. Cyclin G1 interacted directly with MDM2 and promoted the formation of the ARF/MDM2 complex within the initial 24 h period following DNA damage. Furthermore, 48 h after irradiation, accumulation of p53 protein was enhanced in cyclin G1-/- cells compared to wild-type cells. In contrast, in 48 h postirradiated wild-type cells, the cyclin G1-MDM2 complex was found not to be associated with ARF but with the B'alpha subunit of protein phosphatase A. CONCLUSION: These results suggest that cyclin G1 stabilizes and promotes the degradation of p53 protein by associating, respectively, with MDM2 complexes containing ARF and PP2A.

Animals↗
PubMed2003

ACE2 is required for daughter cell-specific G1 delay in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells reproduce by budding to yield a mother cell and a smaller daughter cell. Although both mother and daughter begin G1 simultaneously, the mother cell progresses through G1 more rapidly. Daughter cell G1 delay has long been thought to be due to a requirement for attaining a certain critical cell size before passing the commitment point in the cell cycle known as START. We present an alternative model in which the daughter cell-specific Ace2 transcription factor delays G1 in daughter cells. Deletion of ACE2 produces daughter cells that proceed through G1 at the same rate as mother cells, whereas a mutant Ace2 protein that is not restricted to daughter cells delays G1 equally in both mothers and daughters. The differential in G1 length between mothers and daughters requires the Cln3 G1 cyclin, and CLN3-GFP reporter expression is reduced in daughters in an ACE2-dependent manner. Specific daughter delay elements in the CLN3 promoter are required for normal daughter G1 delay, and these elements bind to an unidentified 127-kDa protein. This DNA-binding activity is enhanced by deletion of ACE2. These results support a model in which daughter cell G1 delay is determined not by cell size but by an intrinsic property of the daughter cell generated by asymmetric cell division.

Base Sequence↗
PubMed2006

Relationship between cyclin G1 and human papilloma virus infection in cervical intraepithelial neoplasia and cervical carcinoma.

OBJECTIVE: To evaluate the overexpression of cyclin G1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma, and the correlation between cyclin G1 and high-risk human papilloma virus (HPV) infection. METHODS: All of the specimens were obtained from the Department of Pathology of China-Japan Friendship Hospital from January 2000 to August 2004. We detected the expression of cyclin G1 with immunohistochemistry, HPV16/18 infection with in situ hybridization, and high-risk HPV infection with Hybrid capture system II (HC-II) in normal group (25 cases), CIN I (48 cases), CIN II (56 cases), CIN III (54 cases), and invasive cervical squamous-cell carcinoma (SCC, 31 cases). RESULTS: The positive rates of cyclin G1 expression in CIN (77.85%) and SCC cervical tissues (87.10%) were significantly higher than normal (8.00%, P < 0.01), and the intensities of cyclin G1 expression in CIN (40.60%) and SCC cervical tissues (61.51%) were significantly higher than normal (2.72%, P < 0.05). The positive rates and intensities of cyclin G1 expression increased gradually with the grade of cervical lesions. High-risk HPV infection rates were higher in CIN and SCC than normal groups (P < 0.05). There was a positive correlation between cyclin G1 expression and high-risk HPV infection detected with HC-II (Kendall's tau-b = 0.316, 0.269, 0.352, and 0.474 in CIN I, CINII, CIN III, and SCC, respectively, P < 0.05). CONCLUSIONS: Cyclin G1 is overexpressed in CIN and SCC. Cyclin G1 may be a biomarker for detecting CIN and SCC. Cyclin G1 may play an important role in the oncogenesis of CIN and SCC by high-risk HPV infection.

Carcinoma, Squamous Cell↗
PubMed1979

Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line.

Growth of a human leukemic T-cell line (CEM C7) in 10(-6) M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasone-treated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechst-amplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.

Cell Cycle↗
PubMed1999

Chondrocyte apoptosis induced by aggrecan G1 domain as a result of decreased cell adhesion.

A major feature of cartilage deterioration during joint injury and disease is aggrecan degradation and the loss of proteoglycan. Most of the degraded fragments are released into the circulatory system except the G1 domain which accumulates locally in the synovial fluid and cartilage because of its hyaluronan-binding ability. In this study, our objective was to investigate the effects of G1 accumulation on chondrocyte function. We chose to mimic the accumulation of G1 domain by developing a method to express G1 in chondrocytes. We transiently and stably expressed aggrecan G1 domain in the cells and tested the effects of G1 in cell adhesion and apoptosis. Overexpression of the G1 construct induced apoptosis in adherent chondrocytes but not in chondrocytes maintained in suspension cultures. Higher levels of G1 expression caused greater reduction in cell-substratum interaction and induced more cell death. The effect was dose dependent. To corroborate our findings, the role of G1 in reducing adhesion and inducing apoptosis was further investigated in fibroblasts. We found that low adherent cultures also had high levels of apoptosis. Our results suggest that G1 induced apoptosis by destabilizing cell-substratum interaction.

3T3 Cells↗
PubMed2002

Golgi localization of Hantaan virus glycoproteins requires coexpression of G1 and G2.

The membrane glycoproteins G1 and G2 of Hantaan virus (HTNV; family Bunyaviridae) are encoded on the M RNA genome segment as a precursor polypeptide that is cotranslationally cleaved by host proteases. G1 and G2 accumulate in the Golgi complex in cells following either virus infection or transfection with M segment cDNA. However, there are conflicting reports in the literature concerning the Golgi targeting of separately expressed G1. To resolve these differences, a series of M segment and G1 coding region cDNA mutants was constructed containing either C-terminal or internal deletions. The intracellular localization of proteins expressed from these constructs was investigated by using confocal microscopy and double-staining immunofluorescence with G1 and G2 specific monoclonal antibodies and antisera specific for markers of the Golgi complex (GM130 and mannosidase II) and of the ER (calnexin). When expressed individually, G1 and G2 were retained in the ER, whereas when coexpressed from separate plasmids, both proteins localized to the Golgi. A construct expressing the whole G1 coding region and the complete signal sequence of G2 (amino acids 1-648 of the precursor) was found to be the minimal G1 protein competent to rescue G2 to the Golgi. This suggests that the G1 cytoplasmic tail including the downstream G2 signal peptide plays an important role in Golgi localization of HTNV glycoproteins. None of the constructs with internal deletions in the cDNA expressed proteins that localized to the Golgi. Our results indicate that the Golgi retention signal of HTNV glycoproteins may depend on the conformation of oligomerized G1 and G2 complex rather than a precise primary amino acid sequence.

Amino Acid Sequence↗
PubMed1996

Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses.

A large placebo-controlled efficacy trial of the rhesus tetravalent (RRV-TV) and serotype G1 monovalent (RRV-S1) rotavirus vaccines was conducted in 1991-1992 at 24 sites across the United States. Protection was 49% and 54% against all diarrhea but 80% and 69% against very severe gastroenteritis for the two vaccines, respectively. Post-vaccination neutralizing antibody titers to the G1 Wa strain, whose VP7 protein is nearly identical to that of the D strain of rotavirus contained in both vaccines, did not correlate with protection against subsequent illness with G1 strains. This result raised the possibility that in infants who developed post-vaccination neutralizing antibody to Wa, breakthrough (i.e., vaccine failure-the occurrence of rotavirus diarrhea after immunization) may have been due to infection by G1 strains that were sufficiently antigenically distinct from the vaccine strain to evade the neutralizing antibodies elicited by vaccination. To test this hypothesis, we initially compared post-vaccination neutralizing antibody titers of vaccinees against Wa and G1 breakthrough strains using sera from subjects who experienced breakthrough. Post-immunization neutralizing antibody titers to Wa elicited by vaccination were significantly (P < 0.001) greater than to the breakthrough strains subsequently obtained from these subjects. This difference did not, however, correlate with lack of protection since similar differences in titer to Wa and breakthrough strains were found using post-vaccination sera from vaccinees who either experienced asymptomatic rotavirus infections or no infections. To determine the genetic basis for these differences, we compared the VP7 gene sequences of Wa with vaccine strain D, 12 G1 breakthrough strains, and 3 G1 control strains isolated during the same trial from placebo recipients. All breakthrough strains were distinct from Wa and D in antigenically important regions throughout the VP7 protein, but these differences were conserved between breakthrough and placebo strains. Furthermore, a comparative analysis of the deduced amino sequences form VP7 genes of G1 rotaviruses from 12 countries indicated that four distinct lineages have evolved. All breakthrough and control strains from the U.S. vaccine trial were in a lineage different from strain D, the serotype G1 vaccine strain. Although the overall results do not support our original hypothesis that immune selection of antigenically distinct escape mutants led to vaccine breakthrough in subjects with a neutralization response to Wa, it cannot be excluded that breakthrough could be partially due to antigenic differences in the VP7 proteins of currently circulating G1 strains.

Amino Acid Sequence↗