The CDK inhibitor, p27Kip1, is required for IL-4 regulation of astrocyte proliferation

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.     

Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We compared the effects of insulin-like growth factor I (IGF-I) and II (IGF-II) on DNA synthesis and proliferation and investigated various signal transduction mechanisms involved in insulin-like growth factor-induced mitogenesis in primary cultures of adult rat hepatocytes. IGF-I stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 75 ng/ml within 4 h of culture. These effects were sensitive to the IGF-I concentration and cell density. Hepatocyte proliferation induced by IGF-I was potentiated by metaproterenol (10(-6) M) as well as by 8-bromo-cAMP, phorbol 12-myristate 13-acetate (PMA; 10(-8) M) and was inhibited by U-73122 (1-(-[[17beta-3-methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-+ ++1Hpyrrol-2,5-dione)), genistein, wortmannin, PD98059 (2'-amino-3'-methoxyflavone) and rapamycin. The IGF-I effect was independent of pertussis toxin (100 ng/ml). IGF-II also dose dependently stimulated hepatocyte DNA synthesis and proliferation with an EC50 of 0.75 ng/ml within 4 h of culture. However, these effects were not dependent on the initial plating density. The stimulatory effects of IGF-II were potentiated by UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) (10(-5) M) and inhibited by phenylephrine, PMA, metaproterenol, 8-bromo-cAMP, PD98059, rapamycin, and pertussis toxin. The IGF-II effects were not affected by genistein, U-73122, and wortmannin. These results suggest that IGF-I and IGF-II rapidly stimulate the DNA synthesis and proliferation of adult rat hepatocytes by separate mechanisms.     

Total hip arthroplasty with use of an acetabular reinforcement ring in patients who have congenital dysplasia of the hip. Results at five to fifteen years

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.     

cAMP inhibits linoleic acid-induced growth by antagonizing p27(kip1) depletion, but not interfering with the extracellular signal-regulated kinase or AP-1 activities

To understand the underlying signaling events of polyunsaturated fatty acid-induced growth, we studied the effect of cAMP on early and delayed growth response events induced by linoleic acid in smooth muscle cells (SMC). cAMP significantly inhibited both basal and linoleic acid-induced DNA synthesis. Linoleic acid-induced early growth response events, such as activation of ERKs, induction of expression of c-fos and jun-B and stimulation of AP-1 activity, however, were not affected by cAMP. In contrast, linoleic acid-induced c-jun expression was blocked by cAMP. cAMP alone stimulated ERKs activation, c-fos and jun-B expression and increased AP-1 activity. Linoleic acid induced depletion of p27kip1 and increased CDK2 activity, events required for G1/S transition. In contrast to early growth response events, linoleic acid-induced G1/S transition signals were significantly inhibited by cAMP. These findings suggest that in addition to inducing immediate early growth response events, linoleic acid mimics growth factors in activating cell cycle events that are associated with G1/S transition in SMC and the negative regulation of linoleic acid-induced growth by cAMP is apparently due to its antagonism with linoleic acid-induced p27kip1 depletion and CDK2 activation.     

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.     

Cholera toxin stimulates type II pneumocyte proliferation by a cyclic AMP-independent mechanism

Cholera toxin (CT) stimulated DNA synthesis by low-density primary cultures of adult rat type II pneumocytes (T2P) in a dose-dependent manner, either in the presence or the absence of serum. In the presence of 1% rat serum, 1 microgram/ml CT also stimulated a 50% increase in cell number over 8 days of incubation (P<0.01); this was in addition to a 2-fold increase in cell number induced by the serum alone (P<0.05). The same dose of CT also elevated intracellular cAMP and the total activity of protein kinase A (both P<0.01), suggesting toxin stimulation of T2P proliferation by a cAMP-dependent mechanism. However, the effect of CT on DNA synthesis could not be mimicked by 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP), nor by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl-cAMP), each tested over a wide range of concentrations. l-Isoproterenol stimulated surfactant secretion by over 5-fold (P<0. 01), but neither the beta-agonist, forskolin nor 3-isobutyl-1-methylxanthine had any significant effect on DNA synthesis. The purified B-subunit of CT stimulated DNA synthesis to the same degree as did the holotoxin, either in the presence or the absence of rat serum. In contrast, the purified A-subunit had no significant effect. These data suggest that cholera toxin stimulates type II pneumocyte proliferation through a mechanism that is independent of cAMP, protein kinase A and toxin-catalyzed ADP-ribosylation.     

Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation

alpha 1-Adrenergic receptor-mediated responses are overwhelming in adult rat hepatocytes. Inversely, beta-responses are predominant over alpha 1-responses in the hepatocytes that have been cultured at a low cell density (10(4) cells/cm2) for 24 h. The insulin-EGF-induced DNA synthesis in the beta-response-dominant hepatocytes was doubled by beta-agonists or cAMP-generating agents added far behind (16-20 h) the addition of insulin/EGF; i.e., immediately before the entry into the S-phase of the cell cycle. Agonists of alpha 1-adrenergic or other Ca2+, mobilising receptors added to the alpha 1-response-dominant hepatocytes increased DNA synthesis only if they were added within 1-2 h after the addition of insulin/EGF, at the early stage of G1-phase. Agonists of "non-dominant" receptors were rather antagonistic to agonists of "dominant" receptors. Thus, agonists of alpha 1-adrenergic (and other Ca2+ mobilising) receptors and agonists of beta-adrenergic (and other cAMP-generating) receptors acted as comitogens in their own particular manners in the presence of growth factors in hepatocytes in which the respective receptor functions were dominant.     

Interaction of retinoblastoma protein and D cyclins during cell-growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells

To explore the regulation and function of D-type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth-inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G1; association of D-type cyclins with cyclin-dependent kinase (cdk) 4 and cdk6; G1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D-type cyclins; and association of p16INK4a, p15INK4b, and p27Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G1 arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G1), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4-fold in treated versus untreated cells. At 5 h, cyclin D-associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4-associated p27Kip1 and, at 9 h, a fourfold increase in cdk6-associated p27Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cells but not their Rb-negative parental Du/145 cells. The data suggest that HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4- and cdk6-associated p27Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state.     

TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.     

Different changes in expression and function of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system

In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA synthesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) with 2% dimethylsulfoxide (DMSO) and 10(-7) mol/L glucagon (a DMSO culture system), as we previously reported. In the present cultures, almost confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10(-7) mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10(-7) mol/L glucagon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO and 10(-7) mol/L glucagon. During stimulation of DNA synthesis, both Cx26 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearance of both Cx26 and Cx32 immunoreactivities and gap junction plaques were observed. Gap junctional intercellular communication (GJIC), as measured by lucifer yellow, which indicated the function of Cx32, decreased markedly from before the onset of the S phase. GJIC measured by propidium iodide, which indicated the function of Cx26, decreased from before the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis, Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment control level. Cx32 immunoreactivity and gap junction plaques also recovered. However, the recovery of GJIC measured by lucifer yellow was later than that of Cx32 expression. These results indicated the different changes of expression and function of Cx26 and Cx32 in the hepatocytes during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap junctions during hepatocyte growth and differentiation in vitro.     

Differential regulation of cell cycle machinery by various antiproliferative agents is linked to macrophage arrest at distinct G1 checkpoints

There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Keratinocyte growth factor and fibroblast growth factor action on DNA synthesis in rat and human hepatocytes: modulation by heparin

Keratinocyte growth factor (KGF/FGF-7) is a member of the fibroblast growth factor (FGF) superfamily. Unlike other members of the family, the biological activity of KGF appears to be restricted to epithelial cells. Here we have tested the activity of KGF, acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) on normal adult rat and human hepatocytes and their modulation by heparin. Although more modest than the growth response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF), recombinant KGF enhanced DNA synthesis in rat hepatocytes by two- to threefold. This stimulation occurred in the absence of serum and of other exogenous growth factors. Addition of heparin inhibited the KGF response. Although basic FGF showed little activity on rat hepatocytes, acidic FGF stimulated DNA synthesis by approximately twofold and was substantially enhanced by heparin. In contrast to rat cells, human hepatocytes consistently failed to respond to KGF, aFGF, or bFGF with or without heparin, under conditions where EGF and HGF stimulated DNA synthesis up to sixfold. These results indicate that KGF is capable of acting as a complete mitogen for rat hepatocytes in culture and that the activity is consistent with expression by these cells of a type II FGF receptor subtype, the KGF receptor. These observations suggest that KGF/aFGF together with proteoglycans may help regulate rat but not human liver growth.     

Dopamine as a novel antimigration and antiproliferative factor of vascular smooth muscle cells through dopamine D1-like receptors

Vascular smooth muscle cell (VSMC) migration and proliferation are believed to play key roles in atherosclerosis. To elucidate the role of vascular dopamine D1-like receptors in atherosclerosis, the effects of dopamine and specific D1-like agonists SKF 38,393 and YM 435 on platelet-derived growth factor (PDGF) BB-mediated VSMC migration and proliferation were studied. We observed that cells stimulated by PDGF-BB (5 ng/mL), showed increased migration and proliferation. These effects were prevented by coincubation with dopamine, SKF 38,393, or YM 435 (1 to 10 mumol/L), and this prevention was reversed by Sch 23,390 (1 to 10 mumol/l), a specific D1-like antagonist. These actions are mimicked by forskolin (1 to 10 mumol/L), a direct activator of adenylate cyclase and 8-bromo-cAMP at 0.1 to 1 mmol/L and are blocked by a specific protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H 89), but not blocked by its negative control, N-[2-(N-formyl)-p-chlorociannamylamino)ethyl]-5-isoquinoline sulfonamide (H 85). PDGF-BB (5 ng/mL)-mediated activation of phospholipase D, protein kinase C, and mitogen activated protein kinase activity were significantly suppressed by coincubation with dopamine. These results suggest that vascular D1-like receptor agonists inhibit migration and proliferation of VSMC, possibly through protein kinase A activation and suppression of activated phospholipase D, protein kinase C, and mitogen-activated protein kinase activity.     

Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints

We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.     

Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.     

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.     

Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.