Analysis of calcium-dependent protein kinase C isoforms in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis.
The profiles of the calcium-dependent protein kinase C (PKC) isozymes alpha, beta, and gamma were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 d, and 60 d) of diethylnitrosamine (DEN)-induced carcinogenesis, using the Solt-Farber "resistant hepatocyte" model (DEN-2-acetylaminofluorene-partial hepatectomy; DEN-AAF-PH), and then related to the presence of focal or nodular gamma-glutamyl transpeptidase (GGT)-positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatography, two peaks, immunologically identified as PKC-alpha and -beta isoforms, were detected in the liver of normal (alpha/beta ratio = 4.0) and treated rats. In DEN-AAF-PH hepatocarcinogenesis an increase in PKC-alpha expression was found after PH (+43 +/- 19% at 48 h, alpha/beta ratio = 5.1; +125 +/- 25% at 96 h, alpha/beta ratio = 4.8), whereas the PKC-beta isoform appeared less significantly modified (+11 +/- 3% at 48 h and +89 +/- 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC-alpha (+96 +/- 20% and +150 +/- 48%, respectively) and PKC-beta isoforms (+158 +/- 41%, alpha/beta ratio = 3.1 and +130 +/- 26%, alpha/beta ratio = 4.4, respectively), occurred along with the appearance of GGT-positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC-alpha and -beta isoform activities similar to those of normal controls. In contrast, saline-AAF-PH-treated rats had downregulation of PKC-alpha after PH (alpha/beta ratio = 1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC-alpha and -beta revealed diffuse positive cytoplasmic signals in GGT-positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt-Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC-alpha isoform may play a specific role in clonal expansion of DEN-initiated hepatocytes after PH.
La Porta
C A
CA
Department of General Physiology and Biochemistry, University of Milan, Italy.
Perletti
G P
GP
Piccinini
F
F
Comolli
R
R
D015073:2-Acetylaminofluorene; D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D002845:Chromatography / Q000379:methods; D004052:Diethylnitrosamine; D017886:Durapatite; D006498:Hepatectomy; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology* / Q000601:surgery; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced* / Q000201:enzymology*; D008297:Male; D008954:Models, Biological; D011493:Protein Kinase C / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013347:Subcellular Fractions / Q000201:enzymology; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D014408:Biomarkers, Tumor; D007527:Isoenzymes; D004052:Diethylnitrosamine; D017886:Durapatite; D015073:2-Acetylaminofluorene; D005723:gamma-Glutamyltransferase; D011493:Protein Kinase C