Transient induction of ornithine decarboxylase mRNA in rat hepatoma cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The contribution of changes in mRNA levels to the induction of ornithine decarboxylase (ODC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat H35 hepatoma cells was analyzed by Northern blot and quantitative dot blot hybridization. ODC mRNA accumulated rapidly in TPA-treated cultures. The increase in message was transient, reaching a peak within about 3 h, then declining to control levels after 18 h. Maximal accumulation of ODC-specific mRNA varied from 3- to 8-fold above control. The TPA dose-response for ODC message accumulation was half-maximal at approximately 0.18 microM TPA. The increase was completely blocked by actinomycin D, suggesting that TPA stimulates the transcription of ODC genes. Inhibition of protein synthesis by cycloheximide (10 micrograms/ml) led to a superinduction of ODC mRNA in the presence of TPA, which suggested that a short-lived protein may be responsible for negative control of ODC expression.

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.

Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.