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C V Clevenger

Publications and source records attributed to C V Clevenger.

At least 37 records · Page 2Linked to original sources
PubMed1996

A functional analysis of protooncogene Vav's role in adult human hematopoiesis.

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

Adult↗
PubMed1995

Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line.

Stimulation of the prolactin receptor (PRLr) with ligand activates multiple kinase cascades. The proximal mediators involved in the activation of the PRL-activated Raf-1 cascade in T-cells, however, remain poorly characterized. The role of one proximal signaling protein, namely p95vav, during PRLr signal transduction was examined in the Nb2 T-cell line. The novel results obtained here indicate that Vav is transiently associated with the PRLr and is necessary for PRL-stimulated proliferation. During PRL stimulation, a rapid and dramatic increase in guanine nucleotide exchange factor (GEF) activity was found to be associated with Vav immunoprecipitates. Concomitantly, an increase in Vav phosphorylation on serine-threonine residues was observed. The Vav-associated GEF activation could be inhibited by staurosporine and calphostin, but not herbimycin, suggesting a modulatory role for phosphorylation at serine-threonine residues. Treatment of Nb2 cells with antisense Vav oligonucleotide ablated Vav expression and blocked PRL-driven proliferation, but failed to inhibit PRL-induced GEF activation within Nb2 lysates. These data indicate that GEF activity may not be intrinsic to Vav as has been previously suggested, but either resides in or is complemented by an associated GEF. Subsequent to the transient activation of associated GEF activity, Vav was found to translocate into the Nb2 cell nucleus. Thus, Vav may utilize two independent mechanisms in T-cells, namely the activation of an associated GEF and direct nuclear internalization, to mediate PRLr signaling.

Animals↗
PubMed1995

Expression of prolactin and prolactin receptor in human breast carcinoma. Evidence for an autocrine/paracrine loop.

The neuroendocrine hormone prolactin is a growth factor required for the proliferation and terminal differentiation of the human breast. These effects are mediated by the prolactin receptor, a member of the growth factor receptor family. Three prolactin receptor isoforms (long, intermediate, and short) have been identified in the rat, which differ in the length of their intracytoplasmic domains. In humans, however, only the long prolactin receptor isoform had been identified previously. The expression of the human intermediate prolactin receptor is demonstrated and preliminary evidence for a human short isoform is presented. Heterogeneous expression of prolactin receptor, at the immunoblot and immunohistochemical levels was observed in breast carcinoma specimens. A statistically significant correlation between prolactin receptor and estrogen receptor expression was noted. An autocrine/paracrine role for prolactin within breast tissues was further examined by performing reverse transcription polymerase chain reaction on RNA isolated from cell lines and clinical specimens with prolactin-specific primers. A 585-bp product was observed and found to be identical to human prolactin. The synthesis of prolactin by breast epithelium was confirmed by in situ hybridization analysis of breast tissues and the detection of bio- and immunoreactive prolactin in breast cancer lines. These analyses indicate that the principal site for prolactin expression within the normal or malignant breast residues within the epithelium. These data indicate that prolactin may participate in an autocrine/paracrine stimulatory loop within breast tissues and suggest a role for this growth factor in the pathogenesis of breast cancer.

Amino Acid Sequence↗
PubMed1994

Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line.

The receptor for prolactin (PRL) is a member of the hematopoietic receptor family that also includes the receptors for interleukins 2-7. PRL is synthesized and is secreted by human T lymphocytes and acts as a lymphokine necessary, but not sufficient, for T lymphocyte progression through the G1 phase of the cell cycle. Although data now indicate that PRL serves an immunomodulatory role in vitro and in vivo, the mechanisms of PRL receptor signal transduction in T cells have not been defined. We demonstrate here that PRL induced the phosphorylation of the p72-74 serine/threonine kinase c-Raf-1 in the PRL-dependent rat T-cell line Nb2. Associated with this inducible phosphorylation of Raf-1 was a concentration- and time-dependent activation of in vitro Raf-1 autokinase and substrate kinase activities, which correlated with the PRL-induced proliferation of Nb2 cells. Co-immunoprecipitation studies revealed association of Raf-1 with PRL receptors in Nb2 cells. These results revealed that all isoforms of the PRL receptor (short, intermediate, and long) are expressed in Nb2 cells and associate with Raf-1. In contrast to the PRL-dependent Nb2 cells, phosphorylation and activation of Raf-1 were constitutive in the Nb2-derived, PRL-independent, T-cell line Sp. These studies demonstrate for the first time an association between the PRL receptor and a serine/threonine kinase affiliated with signal transduction.

Animals↗
PubMed1994

Prolactin as a second messenger for interleukin 2.

The generation of an effective immune response requires the selection and proliferation of antigen-specific lymphocytes. Activation of at least some T lymphocytes involves the sequential stimulation of cell surface receptors, including the T-cell receptor for antigen, the interleukin 2 receptor, and the prolactin receptor. Prolactin signaling in T lymphocytes appears to utilize a classical receptor-mediated kinase cascade and a novel peptide hormone activation pathway involving nuclear translocation.

Antigens↗
PubMed1994

The protein tyrosine kinase P59fyn is associated with prolactin (PRL) receptor and is activated by PRL stimulation of T-lymphocytes.

The clonal expansion of antigen-stimulated T-lymphocytes during an immune response is mediated by several lymphokines. Strong evidence now exists that the neuroendocrine hormone PRL is necessary, but not sufficient, for T-cell proliferation. Little is known, however, of the signal transduction mechanisms of the PRL receptor (PRLR) within T-cells. We demonstrate here that PRL stimulation of the T-cell line Nb2 induced the concentration- and time-dependent activation of the protein tyrosine kinase p59fyn, but not of four other src family protein tyrosine kinases. Activation of fyn was also observed in Concanavalin-A-primed peripheral blood lymphocytes stimulated with PRL and in Nb2 cells incubated with anti-PRLR antibodies. The activation of fyn by PRL stimulation correlated with Nb2 cell proliferation. Immunoblot analysis of anti-fyn and anti-PRLR immune complexes revealed an association between each PRLR isoform and p59fyn. These studies demonstrate for the first time an association between the PRLR and a src family protein tyrosine kinase affiliated with signal transduction.

Animals↗
PubMed1993

Corticotropin-releasing factor mRNA in rat thymus and spleen.

Corticotropin-releasing factor (CRF) initiates stress-induced immunosuppression via the hypothalamic-pituitary-adrenal axis. CRF has also been shown to have direct stimulatory and suppressive effects on immune cells. We have previously detected immunoreactive and bioactive CRF in the rat spleen and thymus. To determine if CRF is synthesized in these tissues, we analyzed rat spleen and thymus for the presence of CRF mRNA. RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction with CRF gene-specific oligonucleotide primers. After Southern blotting and hybridization with an internal CRF gene probe, a product of the expected size was detected in the spleen, thymus, and hypothalamus (positive control) but not in liver or kidney (negative controls), indicating that CRF is synthesized in the spleen and thymus. Furthermore, CRF could be secreted from splenic and thymic adherent cells in culture. Secretion increased severalfold in response to nordihydroguaiaretic acid (NDGA), a lipoxygenase pathway inhibitor, whereas interleukin 1 had no effect, suggesting that regulation of CRF secretion may differ from that in the hypothalamus. CRF mRNA was detected in NDGA-stimulated thymic adherent cells and in both control and NDGA-stimulated splenic nonadherent cells. The finding that CRF is synthesized in the spleen and thymus suggests that locally synthesized "immune" CRF, acting as an autocrine or paracrine cytokine, may have direct regulatory effects on immune function.

Animals↗
PubMed1993

Murine leukemia virus infection in immunocompetent adult mice.

The course of infection of a chronic murine leukemia virus (MuLV), E-55+, which induces thymic lymphoma/leukemia in 100% of mice infected as adults, was followed after inoculation of virus into adult mice which generate both cytotoxic T cell and neutralizing antibody responses (Blank 1976). DNA from the tissues of these infected mice was analyzed using a polymerase chain reaction assay to detect integrated provirus. Infection in adult immunocompetent BALB.K mice results in an acute phase characterized by a high number of cells containing provirus DNA followed by a period in which provirus DNA in most tissues reaches background levels until the development of thymic lymphoma/leukemia at 4-7 months. This pattern of infection is different from what has been observed in immunosuppressed mice in which levels of virus-infected cells remain high throughout the preleukemic and leukemic phases.

Animals↗
PubMed1993

Inhibition of ornithine decarboxylase activity and cell proliferation by ultraviolet B radiation in EGF-stimulated cultured human epidermal keratinocytes.

Irradiation of EGF-stimulated human keratinocytes in vitro with ultraviolet B (UVB) radiation inhibited both ornithine decarboxylase (ODC) activity and cellular proliferation. A dose-dependent reduction in ODC activity occurred in primary cultures of adult facial keratinocytes and neonatal foreskin keratinocytes, and in an SV40-transformed keratinocyte cell line derived from neonatal foreskin. When SV40-transformed keratinocytes were treated with epidermal growth factor (EGF), ODC activity was induced up to 21 times in the absence of ultraviolet radiation. However, pre-treatment with UVB significantly reduced the EGF induction of ODC. For example, 85% less ODC activity was observed in cultures treated with EGF (10 ng/ml) plus 2.5 mJ/cm2 of UVB than cultures treated with EGF alone. To assess the effect of UVB on cell proliferation, normal human epidermal keratinocytes grown in medium containing EGF were irradiated with 5 and 10 mJ/cm2 UVB. At days 3 and 5 post-irradiation a significant (up to 78%) decrease in proliferation was observed. Nevertheless, the mean proportion of viable to dead cells remained similar in both UVB-treated and non-irradiated cell cultures. Northern blot analysis of total RNA isolated from irradiated and sham-irradiated cultures showed that UVB caused approximately a one third reduction in steady-state ODC mRNA levels in EGF-stimulated keratinocyte cultures. Because ODC is an enzyme required for cell proliferation, we propose that the UVB-induced decrease in cell proliferation may result at least in part from UVB inhibition of ODC mRNA accumulation and reduced enzyme activity.

Cell Division↗
PubMed1993

Epidermal growth factor induces ornithine decarboxylase in SV40-immortalized human keratinocytes.

The effect of epidermal growth factor (EGF) on the activity of ornithine decarboxylase (ODC) was evaluated and partially characterized in SV40-transformed, immortalized cultured human keratinocytes. It was observed that the addition of fresh complete medium to confluent cultures resulted in a 10-fold increase in ODC activity. Characterization of this activity using serum-free medium revealed that the increase in ODC activity was primarily due to the presence of EGF (10 ng/ml). A dose-dependent increase in ODC activity was observed when cultures were treated with EGF. Although near maximal induction occurred with EGF concentrations as low as 2.5-10 ng/ml, maximal induction of ODC (25-fold) occurred with an EGF concentration of 50 ng/ml. The peak time for ODC induction was 10 hours following the addition of EGF to keratinocyte cultures. The induction of ODC by EGF was inhibited by pretreatment of cultures with either cycloheximide or actinomycin D, suggesting that both protein synthesis and gene transcription are important in the EGF induction of ODC. EGF significantly increased the steady state levels of ODC mRNA with a peak at 4 hours, preceding the peak in observed enzyme activity as expected. Pretreatment of cultures with retinoic acid (10(-5)-10(-7) M) significantly inhibited the induction of ODC by EGF. Retinoic acid decreased the steady-state levels of ODC mRNA. These data demonstrate that ODC is an enzyme that is induced by EGF in human keratinocytes; this induction probably involves both gene transcription and protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line, Transformed↗
PubMed1992

Requirement for prolactin during cell cycle regulated gene expression in cloned T-lymphocytes.

The neuroendocrine hormone PRL acts as a progression factor during interleukin-2 (IL2) stimulated lymphocyte proliferation. Since the sequential expression of cell cycle regulated genes occurs during this process, we examined the contribution of IL2 and PRL to specific RNA accumulation. Stimulation of the cloned T cell line L2 with IL2 and PRL induced the sequential expression of interferon regulatory factor-1, c-myc, proliferating cell nuclear antigen, thymidine kinase, cyclin B, and histone H3. Stimulation of L2 cells with PRL alone, however, induced only the expression of interferon regulatory factor-1. Depletion of PRL, through the use of an anti-PRL antiserum, inhibited IL2 driven proliferation and the expression of cyclin B and histone H3. These results demonstrate that PRL may regulate T cell proliferation by enhancing the expression of some genes necessary for entry into S-phase.

Animals↗
PubMed1991

Requirement of nuclear prolactin for interleukin-2--stimulated proliferation of T lymphocytes.

Prolactin (PRL) is necessary for the proliferation of cloned T lymphocytes in response to interleukin-2 (IL-2). Translocation of PRL into the nucleus occurs during IL-2--stimulated mitogenesis. Therefore, the function of intranuclear PRL in T cell proliferation was tested. Eukaryotic expression vectors were prepared to express wild-type PRL [PRL(WT)], PRL that lacks the signal sequence for translocation into the endoplasmic reticulum [PRL(ER-)], and chimeric PRL in which the signal peptide was replaced with the sequence that directs the nuclear translocation of the SV40 large T antigen [PRL(NT+)]. Expression of these constructs in a T cell line (Nb2) responsive to PRL and IL-2 resulted in localization of PRL in the extracellular milieu, cytoplasm, or nucleus, respectively. Stimulation with IL-2 alone resulted in a five- to tenfold increase in the incorporation of [3H]thymidine by cells expressing PRL(NT+) or PRL(WT) as compared to PRL(ER-) or the parental Nb2 cells. Only the PRL(NT+) clone proliferated continuously with IL-2 stimulation in the presence of antiserum to PRL. These results demonstrate that nuclear PRL is necessary for IL-2--stimulated proliferation and suggest that a peptide hormone can function in the nucleus without binding to its cell surface receptor.

Amino Acid Sequence↗
PubMed1990

Regulation of interleukin 2-driven T-lymphocyte proliferation by prolactin.

The requirement for prolactin in interleukin 2-driven T-cell proliferation was evaluated. Addition of an anti-prolactin antiserum resulted in the specific inhibition of T-cell proliferation in a time- and dose-dependent manner. Synthesis of prolactin and its mRNA, however, did not occur during interleukin 2 stimulation. Instead, previously internalized prolactin, presumably from fetal bovine serum, appears to serve as the source of prolactin under serum-free conditions. A 7-fold increase in a prolactin receptor occurred as a function of cell cycle progression; accumulation of a 1.6-kilobase prolactin receptor mRNA increased approximately 2-fold. Interleukin 2 stimulation induced the translocation of prolactin into the nucleus and prolactin receptor to the nuclear periphery. These data indicate that extracellular prolactin is requisite for T-cell proliferation and suggest that the effects of prolactin are exerted in the nucleus.

Animals↗
PubMed1990

Interleukin-2 driven nuclear translocation of prolactin in cloned T-lymphocytes.

The nuclear translocation of PRL is demonstrated at the immunofluorescence and electron microscopic (EM) levels in interleukin-2 (IL2)-stimulated cloned T-cells and concanavalin A-stimulated splenocytes. This translocation occurs 2-10 h after IL2 stimulation, and is reversed by the addition of anti-PRL antiserum into the extracellular culture medium. The nuclear localization of PRL in IL2 stimulated T-cells was confirmed by postembedding immunogold EM. The nuclear uptake of PRL after IL2 stimulation was further documented by EM studies using PRL-colloidal gold conjugates. These studies suggest that the intranuclear PRL is translocated from the extracellular medium via an endosomal/lysosomal pathway over a period of several hours. Finally, the requirement for PRL no later than 6 h after IL2 stimulation is demonstrated through the reversible inhibition of T-cell growth with anti-PRL antiserum.

Animals↗
PubMed1988

Flow cytometric analysis of DNA content in Hürthle cell adenomas and carcinomas of the thyroid.

Paraffin-embedded surgical biopsy material from 17 Hürthle cell tumors of the thyroid was examined for DNA content by flow cytometry to assess the diagnostic and prognostic utility of ploidy determinations in these rare tumors. Both adenomas (11 cases) and carcinomas (6 cases) were studied. As a control for methods, ten randomly selected normal autopsy thyroids were analyzed, all of which demonstrated normal diploid DNA content. Among the Hürthle cell tumors, however, aneuploid peaks were present in six adenomas (55%) and in four carcinomas (67%). Similarly, polyploid DNA peaks in the absence of other aneuploid peaks were present in two adenomas and two carcinomas (18% and 33%, respectively). These findings demonstrate the limited value of aneuploidy or polyploidy as diagnostic features for malignancy in Hürthle cell tumors of the thyroid. As for prognosis, there does not appear to be any unfavorable prognostic significance for abnormal DNA content in histologically benign Hürthle cell tumors treated by surgical excision because no metastases or recurrences occurred in this group at a mean disease-free follow-up of 50 +/- 19 months for six aneuploid lesions and 19 +/- 7 months for two polyploid adenomas. Preliminary data suggest that aneuploidy may, however, have an important prognostic value for histologically defined Hürthle cell carcinomas, because the only patient to die from the tumor in this series had an aneuploid Hürthle carcinoma. Thus, the authors' data indicate that the diagnostic utility of DNA content in Hürthle cell tumors is extremely limited and that there does not appear to be any negative prognostic significance for aneuploidy in histologically defined Hürthle cell adenomas.

Adenoma↗
PubMed1987

Quantitative analysis of a nuclear antigen in interphase and mitotic cells.

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.

Antigen-Antibody Complex↗