Biomedical subjects

W J Pledger

Publications and source records attributed to W J Pledger.

At least 19 recordsLinked to original sources

PubMed2001

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis.

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3beta protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3beta protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF --> Src --> Stat3 --> Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

3T3 Cells↗

PubMed2001

p27Kip1 regulates T cell proliferation.

Our studies addressed the mechanism by which serum acts in conjunction with T cell receptor (TCR) agonists to promote the proliferation of primary splenic T cells. When added to resting splenocytes, TCR agonists initiated G(0)/G(1) traverse and activated cyclin D3-cdk6 complexes in a serum-independent manner. On the other hand, both TCR agonists and 10% serum were required for the activation of cyclin E-cdk2 and cyclin A-cdk2 complexes and the entry of cells into S phase. Serum facilitated cdk2 activation by maximizing the extent and extending the duration of the TCR-initiated down-regulation of the cdk2 inhibitor, p27(Kip1). Although p27(Kip1) levels were reduced (albeit submaximally) in cells stimulated in serum-deficient medium, nearly all of the cdk2 complexes in these cells contained p27(Kip1). In contrast, in cells receiving TCR agonist and 10% serum, little if any p27(Kip1) was present in cyclin-cdk2 complexes. Unlike wild-type splenocytes, p27(Kip1)-null splenocytes did not require serum for cdk2 activation or S phase entry whereas loss of the related cdk2 inhibitor, p21(Cip1), did not override the serum dependence of these responses. We also found that cdk2 activation was both necessary and sufficient for maximal expression of cdk2 protein. These studies provide a mechanistic basis for the serum dependence of T cell mitogenesis.

Animals↗

PubMed2001

Interdependence of cdk2 activation and interleukin-2Ralpha accumulation in T cells.

We have shown previously that serum promotes T cell proliferation by acting with T cell receptor (TCR) agonists to efficiently down-regulate p27(Kip1) and activate cdk2-containing complexes. In the studies described here, the effect of serum on the expression of the alpha subunit of the interleukin-2 receptor (IL-2Ralpha) was examined. We found that serum was required for maximal and sustained IL-2Ralpha protein expression and consequent IL-2 signaling in TCR-activated splenocytes. Serum had no effect on IL-2Ralpha mRNA levels and thus modulates IL-2Ralpha expression post-transcriptionally. Unlike wild-type splenocytes, splenocytes exhibiting serum-independent cdk2 activation due to loss of p27(Kip1) efficiently expressed IL-2Ralpha in serum-deficient medium. Conversely, serum did not promote IL-2Ralpha accumulation in conditions in which cdk2 activity was blocked. These findings demonstrate that cdk2 activation is necessary and sufficient for IL-2Ralpha accumulation in TCR-stimulated splenocytes. On the other hand, IL-2 signaling was required (at least in part) for cdk2 activation in these cells. Thus, cdk2 activation, IL-2Ralpha expression, and IL-2 signaling are interdependent events, and we suggest that this feed-forward regulatory loop plays a key role in T cell mitogenesis.

Animals↗

PubMed2001

Permanent growth arrest in irradiated human fibroblasts.

Exposure of human fibroblasts to doses of ionizing radiation sufficient to cause a permanent growth arrest repressed the expression of genes induced late during G(0)/G(1)-phase traverse, including both cyclin A and cyclin E. In addition, radiation prevented the cell cycle-dependent activation of cyclin D1-associated kinase activity and the subsequent phosphorylation of the RB tumor suppressor protein. Exposure to radiation did not alter the cellular levels of cyclin D1 protein, nor did it alter the formation of cyclin D1-CDK4 complexes. Surprisingly, the repression of cyclin D1-associated kinase activity in damaged mitogen-stimulated quiescent cells could not be accounted for by a relative increase in the association of CDKN1A (also known as p21(Cip1)) with cyclin D1 complexes, nor was cyclin D1 activity targeted by increased levels of CDKN1A in irradiated, logarithmically growing cultures under conditions where cyclin A activity was acutely repressed. Therefore, a radiation-induced permanent growth arrest is mediated by pathways that are distinct from those that cause cell cycle delay in damaged cells involving repression of cyclin-dependent kinase activity by CDKN1A.

Cell Division↗

PubMed2000

Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling.

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully defined. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse fibroblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the findings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These findings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse fibroblasts.

3T3 Cells↗

PubMed2000

Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model.

Upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and subsequent cell growth arrest or senescence is one mechanism by which normal cells are believed to respond to stress induced by the constitutively activated GTPase Ras. We hypothesize that in the absence of p21, the onset of Ras-dependent oncogenesis is accelerated. To test this hypothesis, we crossed MMTV/v-Ha-ras transgenic mice into a p21-deficient background. By 63 days of age, all 8 ras/p21-/- mice developed either malignant (mammary and/or salivary adenocarcinomas) or benign (Harderian hyperplasia) tumors. In contrast, by the same age, only one out of nine of the ras/p21+/+ mice developed a tumor. Furthermore, by 94 days of age, half of the ras/p21-/- mice, but none of the ras/p21+/+ mice, developed mammary tumors. p21-deficiency also accelerated the development of salivary (T50=66 days for ras/p21-/- vs T50=136 days for ras/p21+/+) and Harderian (T50=52 days for ras/p21-/- vs T50>221 days for ras/p21+/+) tumors. Furthermore, two out of the eight ras/p21-/- mice had metastatic lesions, one in its lungs, the other in its abdomen. None of the nine ras/p21+/+ mice had metastatic lesions. By 4 months of age, the mammary tumor multiplicity was 10-fold greater in ras/p21-/- (average 3.40 tumors/mouse) than in ras/p21+/+ (average 0.33 tumor/mouse) mice. However, once the tumors appeared, their growth rate, apoptosis level, and mitotic index were not affected by the loss of p21, suggesting that loss of p21 is critical in early but not late events of Ras oncogenesis. Altogether, the results show that tumor onset in MMTV/v-Ha-ras mice is p21-dependent with loss of p21 associated with earlier tumor appearance and increased tumor multiplicity and aggressiveness.

Adenocarcinoma↗

PubMed2000

PCNA interacts with hHus1/hRad9 in response to DNA damage and replication inhibition.

The hHus1 and several hRad proteins are involved in the control of DNA integrity checkpoints, although the mechanisms underlying these processes are unknown. Using a yeast two-hybrid system to detect protein-protein interactions, we found that human proliferating cell nuclear antigen (PCNA), a protein known to function in both DNA replication and repair, interacts with the human checkpoint-related protein Hus1 (hHus1). In human skin fibroblast cells, exposure to ionizing radiation of hydroxyurea triggers translocation of hHus1 from the cytosol to the nucleus, where it associates with PCNA as well as another checkpoint protein, hRad9. This nuclear translocation and the complex formation or hHus1 with PCNA and hRad9 correlate closely with changes in cell cycle distribution in response to radiation exposure. These results suggest that this multi-protein complex may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

Active Transport, Cell Nucleus↗

PubMed2000

Inhibition of mitogenesis in Balb/c-3T3 cells by Trichostatin A. Multiple alterations in the induction and activation of cyclin-cyclin-dependent kinase complexes.

Trichostatin A (TSA), a global repressor of histone deacetylase activity, inhibits the proliferation of a number of cell types. However, the identification of the mechanisms underlying TSA-mediated growth arrests has remained elusive. In order to resolve in more detail the cellular process modulated during the growth inhibition induced by TSA, we studied the effect of the drug on G(0)/G(1) traverse in mitogen-stimulated quiescent Balb/c-3T3 cells. Cyclin D1 and retinoblastoma proteins were induced following the mitogenic stimulation of both control and TSA-treated cells, and cyclin D1 formed complexes with CDK4 under both conditions. However, cyclin D1-associated kinase was not increased in growth-arrested cells. The lack of cyclin D-associated kinase was paralleled by an accumulation of RB in a hypophosphorylated form, as would be expected. In contrast, p130 became partially phosphorylated, accompanied by a marked increase in p130-dependent E2F DNA binding activity and a partial release of free E2F-4. Despite the presence of E2F complexes not bound to pocket proteins, late G(1) E2F-dependent gene expression was not observed. The lack of cyclin D1-associated kinase in TSA-treated cultures was potentially due to high levels of the cyclin-dependent inhibitor p27(kip1). However, the modulation of p27(kip1) levels by the deacetylase inhibitor cannot be responsible for the induction of the cell cycle arrest, since the growth of murine embryo fibroblasts deficient in both p27(kip1) and p21(cip1) was also inhibited by TSA. These data support a model in which TSA inhibits very early cell cycle traverse, which, in turn, leads to a decrease in cyclin D1-associated kinase activation and a repression of late cell cycle-dependent events. Alterations in early G(0)/G(1) gene expression accompany the TSA-mediated growth arrest.

3T3 Cells↗

PubMed2000

Adhesion to fibronectin via beta1 integrins regulates p27kip1 levels and contributes to cell adhesion mediated drug resistance (CAM-DR).

The tumor cell environment may influence drug response through interactions with the extracellular matrix (ECM). We recently reported that adhesion of myeloma cells to fibronectin (FN) via beta1 integrins is associated with a cell adhesion mediated drug resistance (CAM-DR). Activation of beta1 integrins is known to influence both apoptosis and cell growth. We hypothesized that the FN mediated cytoprotection may be in part due to perturbations in cell cycle progression. In this report we demonstrate that adhesion of myeloma cells to FN results in a G1 arrest associated with increased p27kip1 protein levels and inhibition of cyclin A and E associated kinase activity. Disruption of cells from FN adhesion resulted in a rapid recruitment of cells into S phase, a decrease in p27kip1 levels, and reversion to a drug sensitive phenotype. Treatment of cells with p27Kip1 antisense oligonucleotides did not affect FN adhesion; however, p27Kip1 protein levels were reduced and cells became sensitive to cytotoxic drugs. These studies demonstrate that beta1 mediated adhesion of myeloma cells to FN regulates p27kip1 levels and that p27kip1 levels are causally related to CAM-DR. Disruption of beta1 integrin mediated FN adhesion may represent a potential target for the potentiation of drug induced apoptosis.

CDC2-CDC28 Kinases↗

PubMed2000

Activation of Stat3 preassembled with platelet-derived growth factor beta receptors requires Src kinase activity.

Members of the STAT family of transcriptional regulators modulate the expression of a variety of gene products that promote cell proliferation, survival and transformation. Although initially identified as mediators of cytokine signaling, the STAT proteins are also activated by, and thus may contribute to the actions of, polypeptide growth factors. To define the mechanism by which these factors activate STATs, we examined the process of Stat3 activation in Balb/c-3T3 fibroblasts treated with platelet-derived growth factor (PDGF). As STATs are activated by tyrosine phosphorylation, and as PDGF receptors are ligand-activated tyrosine kinases, we considered the possibility that Stat3 interacts with and is phosphorylated by PDGF receptors. We find that Stat3 associates with PDGF beta receptors in both the presence and, surprisingly, the absence of PDGF. Moreover, Stat3 was phosphorylated on tyrosine in PDGF beta receptor immunoprecipitates of PDGF-treated but not untreated cells. Although required, receptor activation was insufficient for Stat3 activation. When added to cells in combination with a pharmacologic agent (PD180970) that specifically inhibits the activity of Src family tyrosine kinases, PDGF did not activate Stat3 as monitored by electrophoretic mobility shift assay. PD180970 did not affect MAPK activation by PDGF or the JAK-dependent activation of Stat3 by interleukin-6. The necessity of Src activity for Stat3 activation by PDGF was further evidenced by data showing the presence of Src in complexes containing both Stat3 and PDGF beta receptors in PDGF-treated cells. These results suggest a novel mechanism of STAT activation in which inactive Stat3 pre-assembles with inactive PDGF receptors, and in response to ligand binding and in a manner dependent on Src kinase activity, is rapidly phosphorylated and activated. Additional data demonstrate that Src kinase activity is also required for PDGF stimulation of DNA synthesis in density-arrested cells.

3T3 Cells↗

PubMed2000

Density-dependent growth inhibition of fibroblasts ectopically expressing p27(kip1).

The cyclin/cyclin-dependent kinase (cdk) inhibitor p27(kip1) is thought to be responsible for the onset and maintenance of the quiescent state. It is possible, however, that cells respond differently to p27(kip1) in different conditions, and using a BALB/c-3T3 cell line (termed p27-47) that inducibly expresses high levels of this protein, we show that the effect of p27(kip1) on cell cycle traverse is determined by cell density. We found that ectopic expression of p27(kip1) blocked the proliferation of p27-47 cells at high density but had little effect on the growth of cells at low density whether exponentially cycling or stimulated from quiescence. Regardless of cell density, the activities of cdk4 and cdk2 were markedly repressed by p27(kip1) expression, as was the cdk4-dependent dissociation of E2F4/p130 complexes. Infection of cells with SV40, a DNA tumor virus known to abrogate formation of p130- and Rb-containing complexes, allowed dense cultures to proliferate in the presence of supraphysiological amounts of p27(kip1) but did not stimulate cell cycle traverse when cultures were cotreated with the potent cdk2 inhibitor roscovitine. Our data suggest that residual levels of cyclin/cdk activity persist in p27(kip1)-expressing p27-47 cells and are sufficient for the growth of low-density cells and of high-density cells infected with SV40, and that effective disruption of p130 and/or Rb complexes is obligatory for the proliferation of high-density cultures.

3T3 Cells↗

PubMed2000

Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1).

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).

3T3 Cells↗

PubMed1998

Primary keratinocytes have an adhesion dependent S phase checkpoint that is absent in immortalized cell lines.

In order to understand the mechanism through which loss of anchorage inhibits growth, we have investigated the events that occur in murine keratinocytes upon substratum detachment utilizing both primary cells and established immortalized cell lines. Our data has revealed that while both primary and immortalized cells undergo growth arrest in suspension, the nature of this arrest is markedly different. Primary cells exhibit a growth arrest that is characterized by rapid cessation of DNA synthesis resulting in a static S phase population. In contrast, an immortalized non-tumorgenic cell line, Balb MK, exhibits growth arrest as measured by thymidine incorporation, but does not prevent cells that have entered S phase from continuing into G2/M, and accumulating as a 4N population. In contrast to both primary and MK cells, the tumorigenic SLC-1 cell line did not accumulate in a specific cell cycle interval and were able to undergo continuous growth in suspension. Examination of cyclin A protein and its associated activity revealed that cyclin A protein levels decreased in primary but not MK cells; suggesting the continued presence of cyclin A may allow continued DNA synthesis observed in MK cells. Furthermore, we demonstrate the accumulation of suspension cultured MK cells as a 4N population correlated with the loss of cyclin A/cdk2 kinase activity, which in turn occurred through the accumulation of p27kip1, whereas neither p27kip1 accumulation nor loss of cyclin A activity was observed in SLC-1 cells. Our results clearly reveal that the process of growth inhibition in suspension cultured cells may occur in several forms with distinct characteristics that are dependent on the status of cyclin/cdk complexes and CKI proteins. Tumor derived cells in suspension did not lose cyclin A dependent kinase activity and thus continued to grow and divide.

Animals↗

PubMed1998

The role of cyclin D3-dependent kinase in the phosphorylation of p130 in mouse BALB/c 3T3 fibroblasts.

We have observed that cyclin D3-dependent kinase activity is increased in the late G1 phase in BALB/c 3T3 fibroblasts. The profile of cyclin D3-associated activity closely parallels that of cyclin D1, which is also induced after mitogenic stimulation of quiescent cells. These activities correlate with the appearance of hyperphosphorylated p130, an Rb family member important in regulating E2F-4 and E2F-5 activity in fibroblastic cells. We demonstrated, however, that only the cyclin D3 activity efficiently phosphorylated p130 in an in vitro kinase assay. This apparent specificity was further demonstrated by experiments which demonstrated that cyclin D3 was physically associated with p130 at the times when D3-dependent kinase activity and p130 hyperphosphorylation were observed. Examination of E2F by electrophoretic mobility shift assay revealed that E2F-4 DNA binding activity existed in a p130.E2F complex at times before D3-dependent kinase activity was apparent and in a free E2F-4 complex after D3 activity developed. Thus, our data suggest that cyclin D3 preferentially phosphorylates p130 and is thereby specifically targeted to overcoming growth-suppressive control mediated through p130 pathways.

3T3 Cells↗

PubMed1998

Cyclin D3-associated kinase activity is regulated by p27kip1 in BALB/c 3T3 cells.

We report that cyclin D3/cdk4 kinase activity is regulated by p27(kip1) in BALB/c 3T3 cells. The association of p27(kip1) was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27(kip1)/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27(kip1)-specific antibodies. The association of p27(kip1) with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27(kip1) protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27(kip1) kinase activity was present throughout the cell cycle. Since we found that the p27(kip1) activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27(kip1) and the active cyclin D3 lacking p27(kip1) by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27(kip1)/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27(kip1) has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.

3T3 Cells↗

PubMed1998

STAT3 activation accompanies keratinocyte differentiation.

The signal transducers and activators of transcription (STAT) family of transcription factors has been demonstrated to play key roles in a variety of cell types under conditions that promote differentiation or cell cycle exit. We report our studies of primary murine keratinocytes in which we demonstrate activation of STAT3 during growth arrest and differentiation. In adherent cells, STAT3-specific DNA binding activity was detected in quiescent cultures, down-regulated upon mitogenic stimulation, and found to reaccumulate as cells reentered quiescence. Suspension culturing of proliferating keratinocytes, which induces differentiation, also resulted in induction of STAT3 activity. Furthermore, induction of STAT3 after suspension culturing did not occur in MK cells, an immortalized murine keratinocyte cell line that does not undergo differentiation. Because STAT3 activation in these cells corresponded tightly with the growth status, we examined whether there was a relationship between the cell cycle machinery and STAT3 activation by inhibiting p27kip1 accumulation, which is observed during growth arrest, with antisense oligonucleotides and by using keratinocytes lacking functional p27kip1. In both cases, there was a loss of STAT3 activation and a concomitant delay in terminal cell cycle withdrawal and in the expression of the differentiation specific marker, keratin 1. Thus, in addition to controlling transcription mediated through E2F, our data demonstrate that alterations in the cell cycle machinery are required for appropriate up-regulation of STAT3 activity that occurs during keratinocyte differentiation.

Animals↗

PubMed1997

p107 and p130 associated cyclin A has altered substrate specificity.

We demonstrate that p107 and p130 immune complexes exhibit kinase activity. We have tested such immune complexes with four substrates commonly utilized to assay Cdk activity, including all three known members of the retinoblastoma family. Immunodepletion revealed this kinase activity could be abolished by removal of either cyclin A or Cdk2 but was unaffected by removal of Cdk4 or any D-type cyclin. The appearance of p107 associated activity followed the accumulation of p107 protein. In contrast, the kinase activity associated with p130 immune complexes became apparent after mid-G1, coincident with p130 hyperphosphorylation. GST-Rb, GST-p107, and GST-p130 (where GST indicates glutathione S-transferase) were equally suitable substrates in p107 and p130 immune complex kinase assays, yielding activity equal to 25% of the cyclin A activity present. The p107 and p130 associated activity was unable to phosphorylate histone H1, suggesting the p107 and p130 associated cyclin A/Cdk2 may represent a distinct pool with a distinct substrate specificity. The p107 and p130 associated activity was released from the immune complexes upon incubation with ATP and Mg2+ and exhibited the same substrate preference observed with the untreated immune complex. Our data suggest that p107 and p130 recognize, or form by association, a distinct pool of cyclin A/Cdk2 that preferentially phosphorylates retinoblastoma family members.

3T3 Cells↗

PubMed1997

The role of p27kip1 in the in vitro differentiation of murine keratinocytes.

We have studied the regulation of cyclins and cyclin-dependent kinase activities during differentiation of primary mouse keratinocytes. Differentiation was induced by placing primary murine keratinocytes into suspension culture, under conditions which prevent cells from attaching to any surface. This treatment induces synthesis of keratin 1, one of the earliest known markers of keratinocyte differentiation, and also results in a profound change in the regulation of G1 and S-phase cyclins and their associated proteins as well as their activities. The placement of cells in suspension culture reduced cyclin A, D1, and E kinase activity within 6 h, accompanied by the cessation of DNA synthesis. K1 mRNA levels were observed to increase after this period, supporting the hypothesis that cell cycle withdrawal precedes the differentiation program. Our data further revealed that the p27kip1 protein level and associated cyclin-dependent kinase inhibitory activity increased when keratinocytes were induced to differentiate. Pretreatment of adherent keratinocytes with p27kip1 antisense oligonucleotides dramatically reduced the accumulation of p27kip1 protein upon subsequent suspension culturing and prevented the onset of differentiation independently of the loss of cyclin-dependent kinase activities. Although antisense oligonucleotide treatment inhibited differentiation, it did not prevent growth arrest. Therefore, the differentiation of primary mouse keratinocytes required a function of Kip other than the inhibition of cyclin-associated activities, and we suggest that this requirement may reflect a novel Rb kinase activity present in Kip immune complexes, which is dependent on the presence of cyclin D3. Thus, the placement of keratinocytes in suspension induces a program that includes loss of cyclin activity, which is linked to terminal growth arrest, and an induction of p27kip1, which is linked to the differentiation program.

Animals↗