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Biomedical subjects

W Wharton

Publications and source records attributed to W Wharton.

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

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

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

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↗
PubMed1998

A methodology report of a randomized prospective clinical trial to assess velopharyngeal function for speech following palatal surgery.

Cleft lip and palate occurs in approximately 1 in every 750 live human births, making it one of the most common congenital malformations. Surgical closure of the palatal cleft does not always result in a velopharyngeal port capable of supporting normal speech. The University of Florida (UF), in collaboration with the University of São Paulo (USP), is engaging in a 5-year prospective, randomized controlled study to compare velopharyngeal function for speech outcomes between patients undergoing palatoplasty for complete unilateral cleft lip and palate performed using the von Langenbeck procedure with intravelar velarplasty and those receiving the Furlow double-reversing Z-plasty palatoplasty. The von Langenbeck procedure was selected as the time-tested standard against which the Furlow procedure could be judged. The Furlow procedure, a relatively new operation, has been reported to yield substantially higher rates of velopharyngeal competency for speech than have most other reported series and theoretically should result in less disturbance to midfacial growth. A total of 608 patients will be entered into one of two age categories. Inclusion of two age groups will allow a comparison of results between patients having surgery before 1 year of age (9-12 months) and patients undergoing surgery at approximately 1.5 years of age (15-18 months). Speech data will be collected and will be available for definitive analysis throughout the last 3 years of the study. Collection of preliminary growth data will require more than 5 years; growth analysis is anticipated to continue until all patients have reached maturity. The Hospital for Research and Rehabilitation of Patients with Cleft Lip and Palate at the University of São Paulo (USP-HPRLLP) in Bauru, Brazil, is uniquely situated for conducting this study. The well-equipped and modern facilities are staffed by well-trained specialists representing all disciplines in cleft-palate management. In addition, an already existing social services network throughout Brazil will ensure excellent follow-up of study cases. The clinical caseload at this institution currently exceeds 22,000, and more than 1200 new cases are added annually. This project represents a unique opportunity to obtain prospective data from a large number of subjects while controlling the variables that have traditionally plagued cleft-palate studies. This study is designed to determine which of the two proposed surgical procedures is superior in constructing a velum capable of affecting velopharyngeal competency for the development of normal speech.

Cleft Palate↗
PubMed1995

Cell cycle constraints on peroxide- and radiation-induced inhibitory checkpoints.

The growth of human skin fibroblasts was reduced in a dose-dependent manner after either treatment with hydrogen peroxide or exposure to ionizing radiation. Serum-starved cells were markedly responsive to the inhibitory properties of large doses of either agent at any time during the first 12-14 h after restimulation. In contrast, when logarithmically growing cells were treated with hydrogen peroxide, a large percentage of G1 cells synchronously traversed S phase in a wave that appeared after a 3-4 h delay, with a population of these cells eventually arresting in late S and G2. An analogous compartment of cells exiting G1 was not obvious when logarithmically growing cells were treated with ionizing radiation alone. However, when irradiated cells were subsequently treated for 4 h with aphidicolin to depress ongoing DNA synthesis to the levels seen in cultures treated with peroxide, a similar pattern of cells synchronously exiting G1 was seen. Therefore, although cells between G0 and S had a marked sensitivity to the inhibitory effects of either peroxide or radiation, logarithmically growing cells in G1 between M and S were far less susceptible to either type of growth inhibition.

Cell Cycle↗
PubMed1995

Serum response element and flanking sequences mediate the synergistic transcriptional activation of c-fos by 12-O-tetradecanoylphorbol-13-acetate and cholera toxin in AKR-2B cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin have been shown previously to act synergistically to stimulate traverse of G0-G1 and entry into S phase in quiescent mouse fibroblasts. These agents also have a synergistic effect on the induction of the endogenous c-fos gene, as well as a transfected reporter construct containing the mouse fos promoter/enhancer region from -397 to +1 cloned upstream of luciferase. A detailed mutational analysis of the c-fos-regulatory region revealed that the synergy between TPA and cholera toxin requires multiple discrete elements, including the binding sites for the serum response factor (-308 to -299), p62/Elk-1 (-316 to -309), on the 5' side of the serum response element, and a CCAAT or E box-binding protein(s) on the 3'-flanking side of the serum response element (-303 to -295 or -297 to -292, respectively). The putative cyclic AMP response element (-65 to -58), shown to be activated in a number of cell types after increases in cyclic AMP levels, mediated an induction by TPA but not by cholera toxin in AKR-2B cells, and was not required for the synergistic transactivation induced by the combination of TPA and cholera toxin.

Animals↗
PubMed1994

Targeted RNA fingerprinting: the cloning of differentially-expressed cDNA fragments enriched for members of the zinc finger gene family.

We have developed and applied a modification of an 'RNA Fingerprinting' protocol previously published by Welsh and McClelland (Nucleic Acids Research 19: 5275-5279 1991) such that cDNA fragments which are both differentially-expressed and enriched for members of a specific gene family can readily be identified. cDNA fragments were amplified with an arbitrary primer initially used in the reverse transcription reaction in combination with a member of a primer set which corresponded to a conserved region within a specific gene family. This technique was used to isolate cDNAs encoding a recently described protein kinase as well as an unknown gene that contained a zinc finger. Several other known genes that contained a zinc finger domain and that were differentially-expressed were also isolated.

Amino Acid Sequence↗
PubMed1993

Multiparameter flow cytometric analysis of the effects of indomethacin on adipocyte differentiation in A31T6 cells.

A31T6 proadipocytes, derived from BALB/c-3T3 clone A31, develop responsiveness to differentiation-promoting agents at density-arrest and differentiate into adipocytes, as determined by the accumulation of cytoplasmic lipid droplets. A flow cytometric assay is being employed to monitor the acquisition of aspects of the differentiated phenotype. In this study, the assay is used to monitor both the rate of differentiation, as defined by the appearance of cells containing lipid droplets and the rate of adipocyte maturation, which involves measurement of increases in cytoplasmic lipid in cells already committed to the differentiation programme. Specifically, we show that: 1) treatment with a combination of indomethacin and dexamethasone causes the maximum percentage differentiation in the population, 2) addition of indomethacin in combination with either dexamethasone or insulin increases the rate of differentiation, and 3) indomethacin selectively increases the maturation of adipocytes, measured as an increase in the amount of lipid per cell. The cytometric assay used in these experiments has allowed determination of the effects of indomethacin on aspects of the adipocyte phenotype that cannot be measured by standard techniques.

Adipose Tissue↗
PubMed1992

Cholera toxin potentiates TPA-induced mitogenesis and c-fos expression in BALB/c-3T3-derived proadipocytes.

Treatment of quiescent density-arrested A31T6 proadipocytes with medium supplemented with either 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or cholera toxin alone did not stimulate G0/G1 traverse and initiation of DNA synthesis. Combinations of either TPA and cholera toxin or insulin and cholera toxin caused a small stimulation of proliferation. Addition of medium supplemented with TPA and insulin caused a marked stimulation of cell cycle traverse which was significantly potentiated by the coaddition of cholera toxin. The actions of cholera toxin were mimicked by forskolin. Expression of c-fos was regulated in a manner that reflected the results of the mitogenic experiments. TPA caused a marked induction of expression, while only a small increase in transcript levels was seen after treatment with cholera toxin. Addition of a combination of cholera toxin and TPA caused a synergistic induction of c-fos expression. The model system described in this paper allows a detailed analysis of the regulation, by independent second messenger systems, of the transcription of a gene in a mitogenically relevant manner.

3T3 Cells↗
PubMed1992

Differentiation of A31T6 proadipocytes to adipocytes: a flow cytometric analysis.

A flow cytometric assay has been developed which provides precise, quantitative information on the accumulation of cytoplasmic triglycerides in individual A31T6 proadipocytes as they differentiate into adipocytes. The opportunity to measure multiple optical parameters on a cell-by-cell basis has enabled us to monitor phenotypic aspects of differentiation with a greater level of sensitivity than was previously possible. Using the fluorescent hydrophobic probe, Nile red, we have found that as a cell proceeds along the differentiation pathway, the gold fluorescence signal from the cell increases, reflecting the accumulation of cytoplasmic lipid droplets. The value of the ratio of gold to red fluorescence defines a cell as being differentiated or undifferentiated. The greater resolution afforded by this cytometric method over more conventional approaches has allowed us to determine (1) the presence of an undifferentiated population of cells whose existence is not detected by conventional phase microscopy, (2) that insulin is not required to drive differentiation in this system, though the rate of differentiation is increased when the cells are exposed to insulin in combination with dexamethasone, (3) that exposure to a combination of insulin and dexamethasone results in a lower accumulation of lipid in a cell than does exposure to either agent alone, and (4) that A31T6 cells show the same response to differentiation-promoting agents whether applied at the time of plating or at confluence.

Adipose Tissue↗
PubMed1991

Isolation of cell membrane for epidermal growth factor receptor studies.

The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the purified cell membranes. The membrane purification procedure has been adapted by others in assessing EGF kinase activity and has been used for the purification of cell membranes from other types of cultured cells.

Animals↗
PubMed1990

Complex mitogenic requirements of Na(+)-K(+)-Cl(-)-cotransport-deficient BALB/c-3T3 cells.

The ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) to stimulate mitogenesis in BALB/c-3T3 cells and in a Na+K+Cl(-)-cotransport-defective variant subclone was investigated. This transport variant had previously been reported to be TPA mitogenically nonresponsive (O'Brien and Prettyman: Journal of Cellular Physiology 130:377-381, 1987) since the addition of TPA to the spent medium of density-arrested cultures stimulated DNA synthesis in the parent but not the variant cell line. We now report that the addition of TPA plus insulin, either directly to the spent medium or together with fresh medium, stimulated DNA synthesis in both the parent and variant cells to approximately the same extent. The parent and transport-deficient cells differed, however, in their sensitivity to the co-mitogenic effects of insulin or insulin-like growth factors.

Animals↗