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PubMed1988

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases.

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.

Animals↗
PubMed1987

Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2.

We have determined the complete nucleotide sequence of the virion M RNA segment of Uukuniemi virus (Uukuvirus genus, Bunyaviridae) from cloned cDNA. The RNA that encodes the two membrane glycoproteins G1 and G2 is 3231 residues long (mol wt 1.1 X 10(6)). The 5' and 3' ends of the RNA are partially complementary to each other for some 30 bp, enabling the formation of a stable panhandle structure (delta G = -40 kcal/mol) and the circularization of the molecule. The extreme 5' and 3' terminal nucleotides are identical for 10 to 13 residues to those of the M RNA of Punta Toro and Rift Valley fever viruses, two members of the Phlebovirus genus. A single open reading frame comprising 1008 amino acid residues (mol wt 113,588) was found in the mRNA-sense strand between nucleotides 18 and 3042. This probably corresponds to the previously identified 110,000-Da precursor (p110) of G1 and G2. By comparing the partial aminoterminal sequences of purified G1 and G2 with the deduced protein sequence we confirmed that the gene order is NH2-G1-G2-COOH. Both mature G1 and G2 are preceded by a stretch of 17 predominantly hydrophobic amino acids likely to represent the signal sequences. At their COOH-terminal ends, G1 and G2 have a hydrophobic stretch of amino acids, 19 and 27 residues, respectively, that probably anchors the proteins to the lipid bilayer. The sequence indicates that mature G2 is 495 amino acids long (mol wt 54,869), whereas the exact size of G1 is unclear, since the location of the COOH-terminus of G1 is not known. An upper value of 479 amino acids (mol wt 55,181) can, however, be suggested. Both G1 and G2 contain four potential glycosylation sites for Asn-linked glycans and both are unusually rich in cysteines, 6.1% in G1 and 5.4% in G2. Comparison of the amino acid sequence of the M RNA product of Uukuniemi virus with that of Punta Toro and Rift Valley fever viruses showed in both cases a weak homology that was more pronounced for the proteins located at the COOH-terminal end of the precursor. This suggests a distant evolutionary relationship between the Phlebo- and Uukuvirus genera.

Amino Acid Sequence↗
PubMed2001

HLA-G1 co-expression boosts the HLA class I-mediated NK lysis inhibition.

It is now acknowledged that the pattern of HLA-G expression is not restricted to extravillous cytotrophoblast cells, as several studies described HLA-G in HLA class I+ cells, such as thymic epithelial cells, cytokine-activated monocytes and some tumors. In these situations, HLA-G may provide an additional inhibitory signal to escape from NK cell-mediated cytotoxicity. Accordingly, the aim of this study was to define the behavior of HLA-G once it is co-expressed into an HLA-A, -B, -C and -E+ cell line. For this purpose, HLA-G1 cDNA was transfected into an HLA class I+ melanoma cell line which was used as a target towards freshly isolated peripheral blood NK cells. Cytotoxic experiments using either anti-HLA-G1 or anti-HLA-G1 inhibitory receptor mAb show that HLA-G1 boosts the HLA class I-mediated inhibition of polyclonal NK cells through interaction with ILT-2, which appears as the major HLA-G1 inhibitory receptor involved. Nevertheless, HLA-G1 is also able to inhibit the cytolytic activity of an ILT-2- NK clone which otherwise expresses another HLA-G1 inhibitory receptor belonging to the KIR103 gene family. In order to more precisely define the relative role exerted by HLA-G1 versus -E on polyclonal NK cells, antibody-blocking assays were carried out using either anti-HLA class I or anti-CD94/NKG2A. Results demonstrate that in the absence of HLA-G1, the naturally expressed HLA class I-mediated NK inhibition is predominantly exerted by HLA-E through binding with CD94/NKG2A. In contrast, once HLA-G1 is expressed, it becomes the major NK inhibitory ligand.

Adjuvants, Immunologic↗
PubMed2003

Predominant cellular immune response to the cartilage autoantigenic G1 aggrecan in ankylosing spondylitis and rheumatoid arthritis.

OBJECTIVES: Based on their HLA association, both ankylosing spondylitis (AS) and rheumatoid arthritis (RA) seem to be T-cell-driven diseases in which the autoantigens remain to be defined. One possible autoantigen is the G1 domain of aggrecan, the major cartilage proteoglycan. In BALB/c mice immunized with this protein, spondylitis and erosive polyarthritis have been reported. Immune reactivity to the G1 has been described in patients with RA and AS in an earlier study. Using novel and more sensitive techniques and relevant controls we sought to define the role of G1 as an autoantigen more precisely and to extend the specific analyses to the peptide level. METHODS: Peripheral blood (PB) mononuclear cells (MNC) from 47 AS patients, 22 RA patients and 20 healthy normal controls were exposed in vitro for 6 h to the cartilage-derived autoantigens G1, human cartilage (HC) gp-39 and collagen II. Synovial fluid (SF) MNC from seven AS and four RA patients were similarly analysed. Furthermore, PB MNC of 15 AS and 10 RA patients were examined with overlapping 18-mer peptides covering the whole G1 protein to identify the immunodominant epitopes. T cells were stained by monoclonal antibodies directed against the surface markers CD4, CD69 and against the intracellular cytokines interferon-gamma (IFN gamma), tumour necrosis factor-alpha (TNF alpha), interleukin 4 (IL-4) and IL-10. The percentage of reactive T cells was quantified by flow cytometry. RESULTS: After antigen-specific stimulation with the G1 protein, the CD4+ T cells of 30 AS patients (61.7%) and of 12 RA patients (54.5%) secreted significant amounts of IFN gamma and TNF alpha, while, in contrast, only 10% of the normal controls showed a response (P < 0.05). The synovial CD4+ T cells of five AS (71.5%) and of all four RA patients showed antigen-specific responses to the G1. In contrast, stimulation with HC gp-39 and collagen II showed no significant IFN gamma and TNF alpha secretion of MNC in all groups. Several G1-derived T-cell epitopes were identified as immunodominant in PB MNC of AS and RA patients and were partly overlapping. CONCLUSIONS: These data show that a cellular immune response to G1 is present in most AS and RA patients. G1-immunodominant epitopes were identified. The relevance of this finding for the pathogenesis of AS and RA remains to be established.

Adipokines↗
PubMed1999

Transient association of calnexin and calreticulin with newly synthesized G1 and G2 glycoproteins of uukuniemi virus (family Bunyaviridae).

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the Bunyaviridae family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257-266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.

Amino Acid Sequence↗
PubMed1993

A Drosophila G1-specific cyclin E homolog exhibits different modes of expression during embryogenesis.

We have isolated a Drosophila homolog of the human G1-specific cyclin E gene. Cyclin E proteins thus constitute an evolutionarily conserved subfamily of metazoan cyclins. The Drosophila cyclin E gene, DmcycE, encodes two proteins with a common C-terminal region and unique N-terminal regions. Unlike other Drosophila cyclins, DmcycE exhibits a dynamic pattern of expression during development. DmcycE is supplied maternally, but at the completion of the cleavage divisions and prior to mitosis 14, the maternal transcripts are rapidly degraded in all cells except the pole (germ) cells. Two modes of DmcycE expression are observed in the subsequent divisions. During cycles 14, 15 and 16 in non-neural cells, DmcycE mRNA levels show no cell-cycle-associated variation. DmcycE expression in these cells is therefore independent of the cell cycle phase. In contrast, expression in proliferating embryonic peripheral nervous system cells occurs during interphase as a brief pulse that initiates before and overlaps with S phase, demonstrating the presence of a G1 phase in these embryonic neural cell cycles. DmcycE appears not to be expressed in cells that undergo endoreplication cycles during polytenization. The structural homology to human cyclin E, the ability of DmcycE to rescue a G1 cyclin-deficient yeast strain, the presence of multiple PEST sequences characteristic of G1-specific cyclins and expression during G1 phase in proliferating peripheral nervous system cells all argue that Drosophila cyclin E is a G1 cyclin. Constitutive DmcycE expression in embryonic cycles lacking a G1 phase, in contrast to expression during the G1-S phase transition in cycles exhibiting a G1 phase, implicates DmcycE expression in the regulation of the G1 to S phase transition during Drosophila embryogenesis.

Amino Acid Sequence↗
PubMed1983

Physiological and pathological factors influencing bovine immunoglobulin G1 concentration in milk.

Bovine immunoglobulin G1 concentration was determined by radial immunodiffusion in 349 milk samples of uninfected quarters, 95 of infected quarters, and 118 blood serum samples from 42 Holstein-Friesian cows taken at days 30, 150, and 270. In lactation, immunoglobulin G1 concentration in milk was not affected by immunoglobulin G1 concentration in blood serum or location of quarters. The immunoglobulin G1 concentrations increased at the end of lactation and in samples collected from cows beyond the third lactation. Uninfected quarters had a mean immunoglobulin G1 concentration of .46 mg/ml. This was less than means from quarters infected by minor or major pathogens. Quarter infection by Staphylococcus aureus resulted in an increase of immunoglobulin G1 concentration in blood serum (9.22 to 11.3 mg/ml). When Corynebacterium bovis was persistent throughout the lactation, immunoglobulin G1 concentration in blood serum was increased (11.26 mg/ml). There was no correlation between somatic cell count and immunoglobulin G1 concentration in uninfected quarters. There was a slight correlation between bovine serum albumin and immunoglobulin G1 concentration in identical quarters (.23). Infection of quarters increased in varying degrees the correlation between immunoglobulin G1 concentration and bovine serum albumin concentration or somatic cell count in milk.

Animals↗
PubMed1995

Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of p44mapk and p42mapk isoforms in the G1 phase and their inactivation at the G1/S transition.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are rapidly activated in response to mitogenic stimuli. Here we examined the enzymatic activity and phosphorylation state of the individual p44mapk and p42mapk isoforms during early G1 and late G1 phase of the mammalian cell cycle. Release of fibroblast cells from early G1 block was accompanied by a rapid rise in the myelin basic protein (MBP) kinase activity of p44mapk and p42mapk, which declined slowly over several hours to reach negligible values as cells enter S phase. When cells were released from late G1 block, the activity of p44mapk and p42mapk increased transiently, and then rapidly declined to baseline values during G1 to S phase transition. Cells released at the G1/S boundary in a medium lacking growth factors entered S phase in the complete absence of MAP kinase activity. Unlike MAP kinases, the histone H1 kinase activity of p33cdk2 was elevated in late G1 arrested cells and continued to increase during S phase entry. The enzymatic activation of p44mapk and p42mapk in both early G1 and late G1 phase was accompanied by an increase in the phosphothreonine and phosphotyrosine content of the proteins. These findings suggest that the sustained activation of MAP kinases during G1 progression and their inactivation at the G1/S transition are two regulatory processes involved in the mitogenic response to growth factors.

Animals↗
PubMed2000

The continuum model and G1-control of the mammalian cell cycle.

The continuum model of the mammalian division cycle proposes that there are no G1-phase specific controls or events. The G1 phase is simply the time when processes begun in the previous cell cycle are completed. In this review, the continuum model is applied the variability of the G1-phase, the existence of G1-less cells, the ubiquitous G1-phase arrest phenomenon, the effect of over-expressed cyclins on G1-phase length, the statistical variation of the cell cycle, the reports of G1-phase syntheses, the proposed variation in retinoblastoma protein phosphorylation in G1-phase, and the myriad findings put forward to support the G1-control model of the mammalian division cycle. The continuum model is a valid description of the mammalian division cycle.

Animals↗
PubMed1996

The requirements for G1 checkpoint progression of Trypanosoma brucei S 427 clone 1.

Trypanosoma brucei S 427 clone 1 accumulated in G1 when incubated under growth-limiting conditions. Further incubation of the G1-restricted organisms in medium containing 10% fetal bovine serum (FBS) and 2 mM hydroxyurea resulted in their reversible arrest after a G1 checkpoint beyond which serum was not required for progress into and through S. Progress of the G1-restricted T. brucei through the G1 checkpoint was linear and required continuous incubation with exogenous serum growth factors. These were principally low and high density lipoproteins; both lipoproteins triggered G1 progression in a dose- and time-dependent manner whilst their removal by immunoaffinity chromatography severely reduced the capacity of FBS to stimulate G1 progression. Serum-induced progress of T. brucei through G1 was Ca(2+)-independent, but required gene transcription, protein synthesis, and continuous kinase activity that was inhibited by tyrphostin 51 and DAPH 1 which typically inhibit epidermal growth factor receptor protein tyrosine kinase activity. The tyrphostin 51-sensitive catalytic activity was not required for T. brucei protein synthesis, glycolysis, or S phase progression but was required for tyrosine phosphorylation of several polypeptides, none of which was specifically associated with serum-induced G1 progression.

Animals↗
PubMed1996

E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest.

BACKGROUND: The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle. This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130. RESULTS: In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16Ink4, which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21Cip1 or p27Kip1 cannot be bypassed either by inactivation of pRb or overexpression of E2F family members. CONCLUSIONS: These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F-pRb complex. The cyclin E-cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.

Base Sequence↗
PubMed1993

Hexamethylenebisacetamide-induced erythroleukemia cell differentiation involves modulation of events required for cell cycle progression through G1.

Hexamethylenebisacetamide (HMBA), a potent inducer of differentiation of transformed cells such as murine erythroleukemia cells, causes a prolongation of the G1 phase of the cell cycle during which commitment to terminal differentiation is first detected. Removal of HMBA prior to the G1 phase aborts commitment. To further define the relationship between the G1 phase and commitment to differentiation, we used two inhibitors of cell cycle progression: aphidicolin, which blocks cells at the G1/S interphase, and deferoxamine, which blocks cells at an earlier stage during G1. HMBA-induced prolongation of G1 is associated with the accumulation of underphosphorylated retinoblastoma protein, decrease in cyclin A protein levels, and commitment to differentiation. G1 arrest of murine erythroleukemia cells induced by aphidicolin or deferoxamine is not associated with accumulation of under-phosphorylated retinoblastoma protein, suppression of cyclin A protein, or commitment of cells to terminal differentiation. Neither of the cell cycle inhibitors alters the effect of HMBA in inducing the G1-associated changes or commitment to differentiation. Taken together, the present findings indicate that the site of action of HMBA which leads to commitment is in a stage of the G1 phase prior to the point of cell cycle block caused by deferoxamine or aphidicolin. HMBA appears to cause cell differentiation with suppression of cell cycle progression by an action that affects events required for cell progression through G1, including accumulation of underphosphorylated retinoblastoma protein and changes in regulation of cyclin levels.

Acetamides↗
PubMed1995

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

Antineoplastic Agents↗
PubMed1999

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines.

PURPOSE: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. MATERIALS AND METHODS: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age responses. RESULTS: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. CONCLUSIONS: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.

Cell Cycle↗
PubMed2000

The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size.

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.

CDC2 Protein Kinase↗
PubMed1999

Fus3p and Kss1p control G1 arrest in Saccharomyces cerevisiae through a balance of distinct arrest and proliferative functions that operate in parallel with Far1p.

In Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition.

Aspartic Acid Endopeptidases↗
PubMed2000

High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors.

Maternal loss of heterozygosity (LOH) of the 11p15 region and overexpression of the insulin-like growth factor (IGF)-II gene are associated with the malignant phenotype in sporadic adrenocortical tumors. In the imprinted 11p15 region, the p57KIP2 gene is maternally expressed and encodes a cyclin-dependent kinase (CDK) inhibitor involved in G1/S phase of the cell cycle. We hypothesized that maternal LOH in malignant adrenocortical tumors could be responsible for loss of p57KIP2 gene expression and, thus, could favor progression through the cell cycle. We investigated 3 normal adrenals, 31 adrenocortical tumors [11 tumors with normal expression of the IGF-II gene (mainly benign) and 20 with IGF-II gene overexpression (mainly malignant)], and the human adrenocortical tumor cell line NCI H295R for expression of the p57KIP2 gene, G1 cyclins (cyclin D2 and E) and G1 CDK (CDK2, CDK3 and CDK4) protein contents and for kinase activity of G1 cyclin-CDK complexes. The expression of p57KIP2, G1 cyclins, and G1 CDKs in benign tumors was similar to that in normal adrenal tissues, as were kinase activities of G1 cyclin-CDK complexes. By contrast, abrogation of the p57KIP2 gene expression and increased expression of G1 cyclins (cyclin E) and G1 CDKs (CDK2 and CDK4) were associated with high activity of G1 cyclin-CDK complexes in malignant tumors and in the H295R cell line. These data suggest that the p57KIP2 gene might act as a tumor suppressor gene in adrenocortical tumors. Maternal LOH with duplication of the paternal allele or pathological functional imprinting of the 11p15 region are responsible for loss of expression of the p57KIP2 gene and increased expression of the IGF-II gene. Consequently, both events favor cell proliferation in malignant adrenocortical tumors.

Adolescent↗
PubMed2000

Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization.

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.

Adenosine Triphosphate↗