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D Prager

Publications and source records attributed to D Prager.

At least 55 records · Page 3Linked to original sources
PubMed1992

Human insulin-like growth factor I receptor function in pituitary cells is suppressed by a dominant negative mutant.

Hybrid receptors were studied in GC rat pituitary cells overexpressing either wild-type 950Tyr (WT) human insulin-like growth factor I (IGF-I) receptors or mutant human IGF-I receptors truncated at position 952 in the beta subunit transmembrane region (952STOP). 125I-IGF-I binding was increased in both 950Tyr (WT) (14-fold) and truncated human IGF-I receptor (952STOP) stable transfectants (50-fold), when compared to untransfected cells that contained endogenous rat IGF-I receptors. Metabolic cell labeling followed by immunoprecipitation with monoclonal alpha and beta subunit-specific antibodies revealed the presence of hybrid rat/truncated human receptors, truncated transfected human receptors, and WT human IGF-I holotetramers. Both mutant and hybrid receptors were degraded slower than 950Tyr (WT) receptors (> 16 h). Despite their markedly increased ligand binding and prolonged receptor half-life, 952STOP transfectants failed to transduce the IGF-I signal to suppress growth hormone (GH). Also, they neither underwent autophosphorylation nor phosphorylated endogenous proteins. The expected suppression of GH by endogenous rat IGF-I receptors was completely abrogated in 952STOP transfectants (P < 0.001 compared to untransfected cells). Mutant 952STOP cells were therefore completely devoid of biological signaling to GH despite the presence of endogenous rat IGF-I receptors. Thus mutant IGF-I receptors block ligand-mediated endogenous rat IGF-I signaling by functioning as a dominant negative forming nonfunctional human/rat hybrid receptors. Defective IGF-I receptors may function therefore as dominant negative phenotypes which suppress normal receptor responses in pituitary cells.

Animals↗
PubMed1992

Rat somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I receptor.

The anterior pituitary contains insulin-like growth factor-II (IGF-II) and expresses abundant IGF-II-binding sites, but the IGF-II signaling pathway in the pituitary has not been defined. We, therefore, tested the effects of recombinant human IGF-II on pituitary function by assessing the GH responsiveness of the primary rat somatotroph to IGF-II. IGF-II (3.3 nM) suppressed GH secretion by 50%, similar to the effect elicited by equimolar doses of IGF-I. In contrast, a low concentration of IGF-II (0.2 nM) did not attenuate GH secretion, while a similar IGF-I dose was sufficient to produce 50% inhibition of basal GH secretion. Fifty percent competition for [125I]IGF-I binding by IGF-I and IGF-II in GC rat pituitary cells demonstrated a 14-fold lesser affinity of the IGF-II ligand for the IGF-I receptor compared to IGF-I; therefore, the binding affinity of IGF-II for the IGF-I receptor correlates with the concentration of IGF-II required for 50% GH inhibition. Transfected GH-secreting cell lines derived from GC cells overexpressing intact human IGF-I receptors exhibited enhanced responsiveness to IGF-II. In contrast, cells transfected with a truncated IGF-I receptor cDNA lacking the cytoplasmic receptor beta-subunit (952STOP) failed to transduce the IGF-II signal, indicating that functional IGF-I receptors are required for IGF-II signaling. In addition, a mutant IGF ligand, [Leu27]IGF-II, which selectively exhibits high affinity for the type II receptor, but only minimal binding to the IGF-I receptor, did not attenuate GH secretion. However, the analog [Arg54,Arg55]IGF-II, which exhibits high affinity to the IGF-I receptor, but no binding to the type II receptor, appropriately suppressed GH secretion. This unique model of somatotroph signaling provides evidence for IGF-II regulation of polypeptide hormone secretion mediated by the IGF-I receptor.

Animals↗
PubMed1991

Binding and action of insulin-like growth factor I in pituitary tumor cells.

Insulin-like growth factor I (IGF-I), a target hormone mediating most of the growth effects of GH, suppresses GH gene expression in a feedback regulatory loop, which may be either endocrine or paracrine in nature. Although IGF-I has been shown to directly attenuate GH gene transcription, the relationship of IGF-I binding and action in the somatotroph cell remains unclear. Therefore, IGF-I binding and action were compared in two different pituitary cell lines both secreting GH. Recombinant human IGF-I attenuated GH secretion in GC cells by up to 70% after 48 h in a dose-dependent manner. Surprisingly, IGF-I failed to suppress GH secretion in GH3 cells, a clonally related pituitary cell line. Binding studies showed that although the KD for IGF-I was similar in both cell types, GC cells contain 3-fold more IGF-I binding sites compared to GH3 cells, possibly explaining their resistance to IGF-I action. Nevertheless, both cell lines possessed abundant and similar binding sites for insulin. These results imply that the IGF-I signal for GH gene regulation is receptor-mediated and directly correlated with the number of pituitary IGF-I binding sites. Availability of pituitary IGF-I binding sites may therefore be important in determining the level of GH expression by the somatotroph.

Animals↗
PubMed1991

Insulin-like growth factor-I (IGF-I) attenuation of growth hormone is enhanced by overexpression of pituitary IGF-I receptors.

Insulin-like growth factor-I (IGF-I) attenuates GH gene expression by a receptor-mediated mechanism in pituitary cells. We, therefore, isolated neomycin-resistant stable GC cell transfectants over-expressing human IGF-I receptor cDNA (IGFIR-cDNA) cloned in an Rous sarcoma virus-directed expression vector. A transfection control contained the IGFIR-cDNA cloned in the reverse orientation. Southern analysis confirmed incorporation of human IGFIR-cDNA sequences into rat genomic DNA. Immunoprecipitation of metabolically labeled [35S]methionine stably transfected cells revealed a 200-kDa human IGF-I receptor precursor protein. Growth rate and basal GH secretion were not altered in transfected cells. Although transfected and control cells had a similar Kd for IGF-I binding (0.43 and 0.40 nM, respectively), IGF-I-binding sites were induced 17-fold (384,000 vs. 22,000 sites/cell). Treatment of cells with IGF-I (6.5 nM) maximally attenuated GH secretion by 80% compared to 40% attenuation in control cells (P less than 0.0001). Maximal suppression of GH in transfectants occurred within 15 h of treatment, and GH secretion by control cells was only maximally suppressed after 42 h. The ED50 of IGF-I suppression of GH secretion in transfectants after 15 h was 0.5 nM. These results demonstrate that transfectants overexpressing human IGF-I receptor are hyperresponsive to exogenous IGF-I. These data indicate that IGF-I receptor number plays an important role in mediating the signal transduction of IGF-I to the GH gene.

Animals↗
PubMed1990

An insulin-induced DNA-binding protein for the human growth hormone gene.

The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced DNA-binding protein was identified. This protein binds to two upstream overlapping oligonucleotide sequences. Binding activity is present at low levels in unstimulated CHO cells and is stimulated by insulin treatment with a time course suggesting that protein synthesis is required. Incubation of the cells with cycloheximide and puromycin confirmed that de novo protein synthesis is necessary for the increased binding activity. Competition with excess unlabeled specific competitor oligonucleotides prevented binding, while unrelated similar-sized oligonucleotides failed to compete for binding, indicating that the observed DNA-protein complex formation is specific. A protein of approximately 70-80 kD was detected by gradient gel electrophoresis. In conclusion, insulin-mediated DNA-protein binding has been identified on the upstream hGH promoter, suggesting a trans-active role for insulin in mediating polypeptide hormone gene expression.

Animals↗
PubMed1988

Insulin regulates expression of the human growth hormone gene in transfected cells.

Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.

Dose-Response Relationship, Drug↗
PubMed1988

Insulin regulates prolactin secretion and messenger ribonucleic acid levels in pituitary cells.

The regulation of PRL production in GH3 rat pituitary tumor cells and normal rat pituitary cells exposed to insulin was studied. Cells were treated with semisynthetic human insulin for up to 5 days. Insulin (0.7 nM) stimulated PRL production by 30%, and cortisol (100 nM) suppressed it by 80% in GH3 cells. Insulin (3.5 nM) partially blocked the suppression of PRL secretion induced by up to 100 nM cortisol. Equimolar doses of insulin-like growth factor I failed to consistently stimulate either basal PRL secretion into the medium or cortisol-suppressed PRL in GH3 cells. A 65 nM concentration of insulin-like growth factor I was required to stimulate basal PRL secretion in GH3 cells. Relative levels of PRL mRNA sequences in both GH3 cells and normal rat pituitary cells were stimulated by insulin. When cells were incubated with cortisol (10 nM), PRL mRNA levels were suppressed to 40% of control values. Simultaneous incubation of cells with insulin (3.5 nM) partially reversed the cortisol-induced suppression of PRL mRNA to 60% of control values. The results show that insulin antagonizes cortisol-induced suppression of PRL. Physiological doses of insulin stimulate PRL production and PRL mRNA levels, suggesting a direct effect of insulin on either PRL gene transcription or stabilization of PRL mRNA in these cells.

Animals↗
PubMed1987

Spontaneous intravenous catheter fracture and embolization from an implanted venous access port and analysis by scanning electron microscopy.

A spontaneous fracture occurred in the midportion of a catheter that was indwelling in the left subclavian vein and embolized to the pulmonary artery. Scanning electron microscopy studies were performed upon the fractured catheter. Possible reasons for the fracture are discussed. Sudden chest pain in a patient with an implanted catheter may be the first clinical manifestation of an embolized catheter.

Adult↗
PubMed1987

Prolonging tamoxifen therapy for primary breast cancer. Findings from the National Surgical Adjuvant Breast and Bowel Project clinical trial.

OBJECTIVE: To determine whether prolonging the duration of tamoxifen administration beyond the cessation of a combined chemotherapy regimen benefits patients with primary breast cancer with positive findings in axillary nodes who benefit initially from the combined regimen. DESIGN: Nonrandomized, nonconcurrent cohort study. SETTING: National Surgical Adjuvant Breast and Bowel Project, conducted in 68 institutions in North America. PATIENTS: Women were included if they had breast cancer with positive nodes and were aged 49 years or less with both estrogen and progesterone receptor levels of 10 fmol or more, aged 50 to 59 years with progesterone receptor levels of 10 fmol or more, or aged 60 to 69 years. Two cohorts were compared: patients who were randomly assigned to the tamoxifen arm of the adjuvant chemotherapy trial (randomized patients) and women who were added to this arm after randomization had ceased (registered patients). Three hundred seventy-seven women in each group who were disease free at the end of the initial 2-year treatment period were followed for an additional 3 years. INTERVENTIONS: All received melphalan, fluorouracil, and tamoxifen (10 mg twice daily by mouth) for 2 years. Registered patients (but not randomized patients) were offered tamoxifen for a third year after the initial 2-year treatment period, and 273 (72%) agreed. MEASUREMENTS AND MAIN RESULTS: Women receiving a third year of tamoxifen had a better disease-free survival rate (odds ratio, 1.54; 95% confidence interval, 1.14 to 2.07; p = 0.004) and survival rate (odds ratio, 1.56; 95% Cl, 1.02 to 2.37; p = 0.04) through their fifth postoperative year. Women aged 50 years or more benefited, but those aged 49 years or less did not. CONCLUSIONS: The benefit of tamoxifen given to tamoxifen-responsive patients in conjunction with melphalan and fluorouracil appears to be enhanced when the tamoxifen treatment is continued beyond cessation of treatment with these agents.

Actuarial Analysis↗
PubMed1986

Single, sequential, and multiple alkylating agent therapy for multiple myeloma: a CALGB Study.

Four intravenous (IV) alkylating agent regimens were tested in 615 previously untreated patients with multiple myeloma. Patients were randomized to receive melphalan, cyclophosphamide, and carmustine in combination (MCBP), sequentially (Seq-MCBP), or in combination with doxorubicin (MCBPA). The fourth group received IV melphalan (MP) as the only alkylating agent. All groups received a tapering dose of prednisone. Toxicity was similar for all regimens although the nadir of cytopenia was reached more quickly for the regime including melphalan only. Response as measured by reduction in myeloma protein or other parameters were similar for the four treatments. Survival was significantly poorer for the group receiving the alkylating agents in sequence. The survival of high tumor cell load patients who were azotemic was better in the groups treated with IV MP or with the combination of IV MCBP. In view of the simplicity and probable cost savings attached to single-agent treatment, a melphalan/prednisone regimen should be considered as initial therapy for all patients with myeloma.

Adult↗
PubMed1981

Treatment of primary breast cancer with chemotherapy and tamoxifen.

We studied the possibility that the addition of tamoxifen to L-phenylalanine mustard combined with 5-fluorouracil enhances the benefit from the latter two drugs that has been observed in women with primary breast cancer and positive axillary nodes. Recurrence of disease was reduced at two years in patients given the three-drug regimen whose tumor estrogen-receptor levels were greater than or equal to 10 fmol. Among patients greater than or equal to 50 years old treatment failure was significantly reduced (P less than 0.001): by 51 per cent in those with one to three positive nodes and by 64 per cent in those with four or more. Higher receptor levels were associated with a greater probability of disease-free survival. Patients less than or equal to 49 years old were less responsive: those with one to three positive nodes received no benefit from tamoxifen at any receptor level, whereas those with four or more appeared to have reduced treatment failure associated with higher receptor levels. This adjuvant chemotherapy is not indicated in patients less than or equal to 49 years old whose tumor receptor levels are below 10 fmol; there is a suggestion of benefit in patients greater than or equal to 50 years old whose levels are low.

Age Factors↗