Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms by which growth factors are degraded and the role this process plays in the regulation of cell growth are not well understood. Insulin degradation is believed to be mediated by a specific metalloprotease, insulin-degrading enzyme (IDE). We have previously shown that IDE can also degrade transforming growth factor-alpha (TGF alpha), but not epidermal growth factor (EGF), in vitro. This selectivity was surprising, since TGF alpha and EGF are structurally similar and bind to the same receptor with comparable affinities. Using a spectrum of protease inhibitors, we have now analyzed the degradation of TGF alpha, EGF, and insulin by human hepatoma HepG2 cells. The results suggest that bacitracin-sensitive metalloproteases are involved in the degradation of TGF alpha and EGF as well as insulin, and that the degradation of TGF alpha, but not EGF, is mediated in part by IDE. Inhibiting the activity of these metalloproteases decreased growth factor depletion, suggesting that these enzymes play an important role in the control of extracellular growth factor levels. The existence of separate degradative pathways for EGF and TGF alpha may explain how the two factors exert differential effects in some systems, and degradation of TGF alpha by IDE would provide a possible mechanism for interaction between the insulin and TGF alpha/EGF signalling systems.
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PMID:Regulation of insulin, epidermal growth factor, and transforming growth factor-alpha levels by growth factor-degrading enzymes. 184 63

Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.
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PMID:Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. 242 18

A cytosolic insulin-degrading enzyme (Mr = 110,000) was found to be cross-linked to [125I]-insulin in intact human hepatoma cells, HepG2, incubated with the hormone and treated with the bifunctional cross-linker, disuccinimidyl suberate. The labeling of this protein was greatly increased by concurrent treatment of the cells with N-ethylmaleimide, to the extent that the amount of [125I]-insulin cross-linked to the enzyme in these cells was approximately 20 to 50% that cross-linked to the insulin receptor. The labeling of the insulin-degrading enzyme required the prior interaction of [125I]-insulin with its receptor as well as a temperature- and energy-dependent processing of the hormone. The present work therefore supports a role for this protease in the cellular processing of insulin.
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PMID:In vivo association of [125I]-insulin with a cytosolic insulin-degrading enzyme: detection by covalent cross-linking and immunoprecipitation with a monoclonal antibody. 302 82

We previously demonstrated that internalized insulin enters the cytoplasm before accumulating in nuclei of H35 rat hepatoma cells. This finding raises the possibility that insulin may interact with cytosolic proteins in addition to insulin-degrading enzyme (IDE). In the present study, cytosol from H35 hepatoma cells, rat liver or muscle was incubated with A14- or B26-125I-insulin at 4 degrees C for 5-120 min in the absence or presence of 25 micrograms/ml unlabelled insulin. 125I-insulin was cross-linked to cytosolic proteins by disuccinimidyl suberate and analysed by reducing or non-reducing SDS/PAGE and autoradiography. Our results demonstrate the presence of both tissue-specific and common cytosolic proteins which specifically bind insulin. In muscle cytosol, only two proteins of 27 and 110 kDa were specifically labelled with B26-125I-insulin. Seven major bands, of 27, 45, 55, 60, 76, 82 and 110 kDa, were labelled in rat liver cytosol. Detection of cytosolic insulin-binding proteins in H35-cell cytosol was dependent on cell-culture conditions. Labelling in cytosol from serum-deprived cells was decreased or absent compared with cytosol prepared from serum-fed or serum-deprived cells treated with 100 ng/ml insulin for 1 h before preparation of the cytosol, in which six bands, of 32, 41, 45, 55, 82 and 110 kDa, were specifically labelled with B26-125I-insulin. This result suggests that the concentration or binding activity of some cytosolic insulin-binding proteins is rapidly regulated. Labelling of both rat liver and H35 cytosolic insulin-binding proteins was time-dependent, and decreased or disappeared at 120 min in parallel with the degradation of labelled insulin. Fewer bands were specifically labelled with A14-125I-insulin than with B26-125I-insulin. The number of labelled bands observed under reducing and non-reducing conditions was not different in any of the cytosols. The 110 kDa band in all cytosols was identified as IDE by Western-blot analysis; the other proteins did not react with anti-IDE antibody and remain unidentified. 1,10-Phenanthroline (2 mM) increased IDE labelling, but decreased the labelling of 82 and 27 kDa bands. The marked difference in the number of cytosolic insulin-binding proteins in muscle and either H35 cells or liver suggests both that the labelling is specific and that these proteins serve a function and may be involved in some heretofore unknown mechanism of the signalling pathway by which insulin regulates cell growth or differentiation.
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PMID:Demonstration of specific insulin binding to cytosolic proteins in H35 hepatoma cells, rat liver and skeletal muscle. 786 12

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels. 802 92

We recently demonstrated that insulin specifically binds to several cytosolic insulin-binding proteins (CIBPs) including insulin-degrading enzyme (IDE) and CIBP p82 in cytosol isolated from H35 rat hepatoma cells. Insulin binding to these CIBPs was regulated by culture conditions, such as serum or insulin. In the present study, we examined the effect of dexamethasone on insulin binding to CIBPs in H35 cells. When the cells were treated with 100 nM dexamethasone for 24 hrs, insulin binding to IDE and CIBP p82 was decreased by about 50% without decreasing the expression level of IDE. Insulin added with the dexamethasone prevented the steroid's effect. Furthermore, dexamethasone directly blocked insulin binding to CIBPs in isolated cytosol. These results suggest that dexamethasone, directly or as a complex with other proteins, binds to IDE and CIBP p82 and changes their ability to bind insulin, possibly by inducing a conformational change or by blocking insulin binding sites. IDE was recently identified as a receptor accessory factor for androgen and glucocorticoid receptors and plays an important role in the regulation of gene transcriptional responses. Combined with previous reports, our findings suggest IDE and other CIBPs such as CIBP p82 may play a role in the cross-talk between insulin and the signal transduction pathways of steroid hormones.
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PMID:Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82. 857 22

We have investigated the biosynthesis, subcellular location and expression of insulin-degrading enzyme (IDE). a type-I peroxisomal protease, in semi-permeabilized hepatoma cells using pulse-chase experiments, non-denaturing immunoprecipitation protocols and Northern-blot analyses. In HcpG2 cell lysates prepared from cells radiolabelled with Tran[35S]-label, immunoprecipitated IDE was observed immediately after a 5 min pulse and subsequently declined during chase with t1/2 of approx. 33 h. In addition to the 110 kDa IDE protein, a protein of 70 kDa (p70) was identified in radiolabelled immunoprecipitates when using a monoclonal anti-IDE antibody 9B12 under non-denaturing conditions. This same antibody did not recognize p70 on Western blots of whole-cell lysates nor in sequential immunoprecipitates of immunocomplex-bead eluates from anti-IDE immunoprecipitations. Likewise, cross-linking studies performed on intact HepG2 and H35 hepatoma cells in vivo revealed the existence of a hetero-oligomeric complex of 180 kDa in which IDE and p70 were physically associated. Digitonin-permeabilization studies in normal and 35S-labelled HepG2 cells have defined a predominant association of IDE and its associated protein p70 with cytosol (supernatant); only a minor amount of the protein IDE was detected in peroxisomes (cellular pellet). Immunoprecipitation of IDE from 35S-labelled cell lysates of normal and stably transfected Chinese hamster ovary cells overexpressing IDE failed to detect p70. Treatment of HepG2 cells with clofibrate, a peroxisome proliferator, resulted in a dose-dependent increase of the two human IDE transcripts of 3.6 and 3.2 kb. This effect was not accompanied by a similar change at the protein level, nor by a change in the subcellular location of the proteins IDE and p70. Based on these findings we propose that in hepatoma cells: (1) IDE mainly exists in a stable cytoplasmic pool that is unchanged in cells undergoing peroxisomal proliferation; and (2) p70 binding to IDE may serve to maintain the dual cytosolic and peroxisomal pools of IDE in a stable equilibrium.
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PMID:Association of insulin-degrading enzyme with a 70 kDa cytosolic protein in hepatoma cells. 887 Jun 62

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.
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PMID:Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: implications for cell proliferation control. 2036 53