Methotrexate-induced apoptosis in hepatocytes after partial hepatectomy.
To investigate apoptosis induced by methotrexate in hepatocytes in vivo, rats received a single injection of methotrexate immediately after partial hepatectomy and apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and gel electrophoresis of DNA. Characteristic DNA fragmentation was obvious at 2 h and peaked at 4 h after partial hepatectomy with methotrexate injection. TUNEL-positive staining was observed in nuclei and nuclear fragments of hepatocytes in the methotrexate-injected liver (partial hepatectomy with methotrexate), with negligible background staining in the control (partial hepatectomy only) and in the methotrexate-injected normal (normal with methotrexate) rat liver. The involvement of the c-Jun N-terminal kinase (JNK) activator protein 1 (AP-1) pathway and p53 in apoptosis was also examined. The activity of JNK increased at 15 min and peaked at 1 h after partial hepatectomy. This increase was repressed by methotrexate injection. Western blot analysis showed that the levels of c-Fos and c-Jun protein expression, which increased at 1 h after partial hepatectomy, were also reduced by methotrexate. The levels of p53 protein were markedly increased after partial hepatectomy with methotrexate injection. The increase in p53 protein was followed by an up-regulation of p21(WAF1/CIP1) protein at 2 h after partial hepatectomy. These results suggested that the inhibition of the JNK-AP-1 pathway and concurrent up-regulation of p53 and p21(WAF1/CIP1) were involved in hepatocyte apoptosis induced by partial hepatectomy with methotrexate.
Kobayashi
Kaoru
K
Department of Food Science and Nutrition, Nara Women's University, Nara 630, Japan.
Terada
Chieko
C
Tsukamoto
Ikuyo
I
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000187:drug effects / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006498:Hepatectomy; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D048031:JNK Mitogen-Activated Protein Kinases; D008297:Male; D008727:Methotrexate / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000378:metabolism
C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005493:Folic Acid Antagonists; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D016159:Tumor Suppressor Protein p53; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D008727:Methotrexate