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J L Messina

Publications and source records attributed to J L Messina.

53 records · Page 3Linked to original sources
PubMed1992

The regulation of p33 gene expression by insulin and calcium ionophores.

We have previously shown that insulin induces p33 transcription and mRNA levels in serum-deprived rat H4 hepatoma cells. In the current study, the effects of the calcium ionophores A23187 and ionomycin on the regulation of p33 gene expression were examined. When H4 cells were incubated with A23187 or ionomycin (1 microM) for 90-300 min, 700-900% and 400-600% increases in p33 mRNA levels were observed. The effects of ionophore and insulin together on p33 mRNA levels were not additive. Insulin-induced increases in p33 mRNA levels were diminished at low concentrations of extracellular calcium, but were unchanged by the calcium channel blocker verapamil. The chelation of intracellular calcium using 20 and 60 microM quin2-AM resulted in 50% and 90% reductions in insulin-induced p33 mRNA levels. When transcription assays were performed, A23187 treatment for 15-180 min increased p33 transcription 300-400%. The lesser effect of A23187 on transcription compared to that on mRNA levels was also true for insulin treatment. Similar results were obtained using ionomycin. Insulin- and A23187-induced p33 transcription was reduced by quin2-AM to below basal levels. These studies show that calcium ionophores stimulate p33 gene expression and suggest that changes in intracellular calcium can alter insulin's induction of this gene.

Animals↗
PubMed1992

Induction of cytoskeletal gene expression by insulin.

Insulin has rapid pleiotropic effects on cellular metabolism. In certain cell types, insulin can cause morphological changes by inducing rearrangements of cytoskeletal components, but the regulation of cytoskeletal gene expression by insulin has not been previously described. In the present work insulin was found to rapidly, but transiently, increase transcription of the cytoskeletal beta-actin and alpha-tubulin genes in rat H4IIE hepatoma cells. Insulin-induced transcription of beta-actin mRNA was evident within 5 min and was maximal by 10-15 min at 1000% above control levels. beta-Actin transcription was induced at insulin concentrations as low as 5 x 10(-12) M insulin and was maximal at 5 x 10(-9) M. Transcription of the alpha-tubulin gene was also rapidly stimulated by physiological concentrations of insulin, but only to 300-400% above basal levels. For both the beta-actin and alpha-tubulin genes, the induction of transcription was transient, with a return to basal levels by 60-120 min. Transcription of neither the skeletal or cardiac alpha-actin gene nor the beta-tubulin gene was altered by insulin administration. Messenger RNA levels for the beta-actin and alpha-tubulin genes increased, but to a lesser extent than transcription, since these mRNAs were abundant and stable before the transient induction of transcription. Inhibitors of protein synthesis, in the presence or absence of insulin, also acutely stimulated transcription of these genes.

Animals↗
PubMed1991

Inhibition and stimulation of c-myc gene transcription by insulin in rat hepatoma cells. Insulin alters the intragenic pausing of c-myc transcription.

One of insulin's least studied actions is its ability to induce DNA synthesis and cell division. In rat H4IIE hepatoma cells insulin, acting through its own receptor, stimulates cell division. However, little is known about the molecular mechanisms involved in this effect. The proto-oncogene c-myc is a cellular gene which when expressed at abnormal levels is often associated with the process of tumorigenesis. Expression of the normal cellular myc gene may be necessary for growth factor-induced cell cycling. In the present work, insulin was shown to regulate cellular accumulation and transcription of the c-myc gene in rat hepatoma cells. The control of c-myc by insulin was complex, with an initial-induced decrease in c-myc transcription to 50% of control values at 15 and 30 min. This was followed by an increase in transcription of about 3-fold by 60-120 min. Similar to the initial inhibitory effect of insulin, the protein synthesis inhibitors cycloheximide or anisomycin decreased c-myc transcription. However, there was no secondary induction of c-myc transcription by protein synthesis inhibitors. The effects of both insulin and protein synthesis inhibitors were shown to be through alterations in intragenic pausing of transcription of the sense mRNA, not through changes initiation of transcription.

Animals↗
PubMed1990

Interaction of insulin and phorbol esters on the regulation of DNA synthesis in rat hepatoma cells.

Insulin and phorbol esters stimulated DNA synthesis in rat H4 hepatoma cells. Insulin and phorbol ester induction of thymidine incorporation was dose-dependent, with a maximal 4.2- and 3.0-fold increases at concentrations of 1 x 10(-9)M and 1 microM, respectively. Phorbol esters in combination with increasing concentrations of insulin resulted in additive effects, but only at submaximal insulin concentrations. The combination failed to increase thymidine incorporation above the maximal effects produced by insulin alone. When cells were pretreated with phorbol esters for 24h to produce protein kinase-C (PKC) deficiency, basal DNA synthesis was depressed. Pretreatment with phorbol esters abolished the effects of phorbol esters to induce DNA synthesis but did not impair the magnitude of insulin-induced DNA synthesis. Thus, although phorbol ester-activatable PKC-activity was necessary for basal DNA synthesis, it was not necessary for insulin-induction of DNA synthesis in H4 cells.

Animals↗
PubMed1990

Decreased induction of an hepatic mRNA by phorbol esters after insulin desensitization.

Insulin and phorbol esters rapidly induce the transcription and cytoplasmic accumulation of a specific mRNA (p33) in rat hepatoma cells. We have studied the effects of insulin desensitization on the regulation of p33 gene expression by insulin and phorbol esters. Insulin desensitization is associated with down-regulation of the insulin receptor and post-receptor defects. When cells were treated with insulin (5 x 10(-7) M) for 24 h, a greater than 50% reduction in insulin binding was observed and insulin's stimulation of p33 transcription and cytoplasmic mRNA levels was prevented. The induction of p33 gene transcription and mRNA levels by phorbol esters was also decreased. Beta-tubulin gene expression was unaffected by insulin or phorbol esters and the stimulatory effect of dexamethasone on p33 gene expression was not impaired. Since insulin desensitization impaired phorbol esters' induction of p33 gene expression, one intracellular defect in insulin-desensitized cells may include an alteration in protein kinase C-dependent events.

Animals↗
PubMed1990

Insulin's regulation of c-fos gene transcription in hepatoma cells.

In H4IIE rat hepatoma cells insulin interacts with its receptors to induce DNA synthesis and promote cell division. However, the postreceptor events that lead to DNA synthesis and cell division have not been well characterized. Previous studies indicate that insulin can regulate the expression of several genes in H4 cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. In the present work insulin is shown to regulate cellular c-fos mRNA accumulation and the transcription rate of the c-fos gene. Insulin caused a rapid, dose-dependent increase in the cytoplasmic concentration of c-fos mRNA which was maximal by 30 min. Preceding this, a more rapid 6-8 fold increase in transcription of the c-fos gene was observed. Induction of transcription was apparent following only 5 min of insulin addition. This is the most rapid effect of insulin yet demonstrated on the induction of gene expression. Protein synthesis inhibitors (cycloheximide, anisomycin) also induced the transcription of the c-fos gene. However, they stimulated a much greater increase in transcription than did insulin, and followed a different time course of action. The addition of insulin in combination with a protein synthesis inhibitor resulted in no greater increase in c-fos transcription than did the addition of a protein synthesis inhibitor alone. The nonadditivity of H4 cell c-fos gene expression may indicate a similar mode of action by insulin and protein synthesis inhibitors.

Animals↗
PubMed1989

Insulin and dexamethasone regulation of a rat hepatoma messenger ribonucleic acid: insulin has a transcriptional and a posttranscriptional effect.

Experiments were conducted to investigate the actions of glucocorticoids and insulin on the induction of a specific mRNA (p33-mRNA) expressed in rat H4 hepatoma cells. Previous studies have found a 10-fold increase in this mRNA following 60 min of insulin addition. In the present study, dexamethasone (Dex) induced the cellular concentration of p33-mRNA 10- to 15-fold. This effect was time and dose dependent. The effect of Dex could be accounted for by a 10- to 15-fold increase in p33-mRNA transcription. However, insulin administration resulted in only a 3-fold increase in the transcription of p33-mRNA. The insulin induction of transcription was time and dose dependent and was blocked by the addition of alpha-amanitin. There was no increase in the transcription of a control gene, beta-tubulin, by either insulin or Dex. Neither insulin nor Dex altered the stability of p33-mRNA. Since the cellular concentration of p33-mRNA was induced to a greater extent than was transcription, insulin must be regulating at least one other step in the sequence between RNA synthesis and RNA stability.

Animals↗
PubMed1988

Transcriptional regulation of a rat hepatoma gene by insulin and protein kinase-C.

Both insulin and phorbol esters rapidly stimulated the cytoplasmic accumulation of a specific mRNA (designated p33) in a time- and dose-dependent manner in serum-deprived rat H4 hepatoma cells. When cells were pretreated with phorbol esters to produce a deficiency in protein kinase-C, the ability of further phorbol ester addition to stimulate p33 mRNA accumulation was abolished. However, after pretreatment of H4 cells with phorbol esters, insulin still induced cellular p33 mRNA concentrations, but to a lesser degree. The primary effect of phorbol esters was to increase transcription of the p33 gene, and this was abolished after pretreatment with phorbol esters. In previous work, insulin was shown to stimulate p33 gene transcription, but this effect was insufficient to account for the level of insulin-induced p33 mRNA production. The transcriptional effect of insulin was further reduced by phorbol ester pretreatment. Insulin must, therefore, regulate p33 gene expression by at least two pathways, at least one of which may be modulated by protein kinase-C.

Animals↗
PubMed1987

Insulin-mimetic actions of wheat germ agglutinin and concanavalin A on specific mRNA levels.

Insulin has pleiotropic effects on sensitive cells, including the regulation of specific mRNA accumulation initiated by the binding of insulin to its plasma membrane receptor. Lectins, such as wheat germ agglutinin (WGA) and concanavalin A (Con A), are known to be insulin mimetic. It is thought that WGA and Con A interact with the insulin receptor or associated membrane glycoproteins which, when activated, lead to insulin-mimetic responses. We attempted to determine whether WGA and Con A could induce the accumulation of a specific messenger RNA (p33-mRNA). Insulin treatment of H4IIE (H4) hepatoma cells increased the concentration of p33-mRNA within 30 min after addition, with a maximum effect of 10- to 15-fold. WGA and Con A also exhibited time- and dose-dependent stimulatory effects on p33-mRNA accumulation with maximal effects of 30- to 40-fold. The effect of insulin was maximal by 1 h and plateaued thereafter, whereas lectins had maximal effects at 2 h after addition to cell cultures. Insulin, WGA, and Con A did not significantly alter the stability (half-life) of p33-mRNA. The addition of RNA synthesis inhibitors blocked the ability of insulin, WGA, and Con A to induce the amount of p33-mRNA. These data suggest that lectins, as well as insulin, induce the synthesis of p33-mRNA in acutely treated H4 hepatoma cells.

Animals↗
PubMed1987

Positive interaction between insulin and phorbol esters on the regulation of a specific messenger ribonucleic acid in rat hepatoma cells.

With Northern blot analysis, we demonstrate that phorbol 12-myristate 13-acetate (PMA) stimulated P-33 messenger RNA (mRNA)-accumulation in rat hepatoma cells in a time- and concentration-dependent manner similar to insulin and plant lectins. No effect of PMA on P-33 mRNA half-life was detected when mRNA synthesis was inhibited with either actinomycin D or 5.6-dichloro-1-beta-D-ribofuranosyl benzimidazole. The effects of insulin and PMA were additive at submaximal concentrations and no additivity was observed under these conditions at maximal concentrations. Thus PMA has a marked insulin-like effect on the accumulation of P-33 mRNA in rat hepatoma cells.

Animals↗
PubMed1985

The effects of insulin and concanavalin A on the accumulation of a specific mRNA in rat hepatoma cells.

One of insulin's effects is to stimulate specific mRNA synthesis. Treatment of H4IIE hepatoma cells with 0.01-1.0 nM insulin results in a maximum 10-15 fold increase in the accumulation of a specific mRNA (p33-mRNA) as measured with a cloned cDNA. Concanavalin A, a lectin known to mimic many of insulin's effects, also stimulates the accumulation of p33-mRNA. The effects of both insulin and Con A were blocked by the addition of two RNA synthesis inhibitors, actinomycin D or 5,6 dichloro-1-beta-D-ribofuranosyl-benzimidazole. We therefore suggest that insulin and concanavalin A act to stimulate p33-mRNA synthesis.

Animals↗
PubMed1985

Effects of insulin alone on the accumulation of a specific mRNA in rat hepatoma cells.

Experiments were conducted to investigate the action of insulin alone on the induction of a specific mRNA referred to as p33. In 24-h serum-deprived rat liver hepatoma cells (H4) grown in monolayer cultures, insulin induced a 3-fold increase in the levels of p33 mRNA within 30 min, and a maximum 10-fold increase was observed by 1 h. The effects of insulin were evident at concentrations as low as 10(-12) M and were maximal at 0.5-1 X 10(-8) M. The effect of insulin was specific, since the level of mRNA for the histocompatibility complex, H2d, was unaffected by insulin. The increase in p33 mRNA was not due to an effect of insulin on the stabilization of mRNA, since insulin did not alter the half-life of this message. In addition, two different RNA synthesis inhibitors blocked the stimulation of p33 mRNA production by insulin. These data suggest that insulin may specifically stimulate the synthesis of p33 mRNA which results in an increased accumulation of total cellular p33 mRNA.

Animals↗
PubMed1985

Inhibition of insulin receptor phosphorylation by indomethacin.

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the beta-subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the beta-subunit of the solubilized insulin receptor by 60% and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-14C]-methyl-D glucose transport by 50% (P less than 0.01), D-[6-14C]-glucose oxidation by 50% (P less than 0.01), D-[6-14C]-glucose conversion to lipid by 30% (P less than 0.01), and D-[1-14C]-glucose conversion to lipid by 60% (P less than 0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-14C]-methyl-D-glucose transport by 75% (P less than 0.01), D-[6-14C]-glucose oxidation by 20% (P less than 0.05), D-[1-14C]-glucose oxidation by 35% (P less than 0.01), D-[6-14C] glucose conversion to lipid by 25% (P less than 0.01), and D-[1-14C] glucose conversion to lipid by 45% (P less than 0.01). In contrast, insulin binding to its receptor, basal D-[1-14C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1985

Effects of hypophysectomy and GH administration on bovine and human GH binding to rat liver membranes.

Experiments were conducted to investigate the specific binding of highly purified bovine and human growth hormones (bGH and hGH) to purified liver plasma membranes of male rats at various times after hypophysectomy and after the acute intravenous administration of bGH. Liver membranes prepared from hypophysectomized male rats showed a two- to threefold increase in the specific binding of either [125I]iodo-bGH or [125I]iodo-hGH, when compared with membranes prepared from the livers of age-matched normal male rats. The increase in GH binding was apparent within 3 days after hypophysectomy and persisted for a number of weeks after the operation. The increase in GH binding produced by hypophysectomy appeared to be due to an increase in the number of binding sites present on the membranes. The intravenous injection of 200 micrograms of bGH into hypophysectomized male rats 5-60 min before they were killed markedly reduced the ability of liver membranes prepared from these animals to bind [125I]iodo-bGH specifically. This decrease in GH binding seen after the injection of bGH may have been due to the development of a slowly dissociating hormone-binding site complex, which thereby reduced the number of available binding sites. This conclusion is supported by the finding that bGH, which is bound in vitro to isolated liver membranes, dissociates slowly and incompletely in the presence of an excess of unlabeled hormone. Moreover, the degree to which the bound hormone can dissociate appears to depend on the length of time that association is allowed to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Selective lymphadenectomy in patients with Merkel cell (cutaneous neuroendocrine) carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive cutaneous tumor with a propensity for local recurrence, regional and distant metastases. There are no well-defined prognostic factors that predict behavior of this tumor, nor are treatment guidelines well established. METHODS: Staging of patients with a new diagnosis of MCC was attempted using selective lymphadenectomy concurrent with primary excision. Preoperative and intraoperative mapping, excision, and thorough histologic evaluation of the first lymph node draining the tumor primary site [sentinel node] was performed. Patients with tumor metastasis in the sentinel node underwent complete resection of the remainder of the lymph node basin. RESULTS: Twelve patients underwent removal of 22 sentinel nodes. Two patients demonstrated metastatic disease in their sentinel lymph nodes, and complete dissection of the involved nodal basin revealed additional positive nodes. The node-negative patients received no further surgical therapy, with no evidence of recurrent local or regional disease at a maximum of 26 months follow-up (median 10.5 months). CONCLUSIONS: While the data are preliminary and initial follow-up is limited, early results suggest that sentinel lymph node mapping and excision may be a useful adjunct in the treatment of MCC. This technique may identify a population of patients who would benefit from further surgical lymph node excision.

Biopsy↗