p53-independent induction of rat hepatic Mdm2 following administration of phenobarbital and pregnenolone 16alpha-carbonitrile.
Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.
Nelson
David M
DM
Discovery Toxicology, Bristol Myers Squibb Co, Princeton, New Jersey 08543, USA.
Bhaskaran
Vasanthi
V
Foster
William R
WR
Lehman-McKeeman
Lois D
LD
D000284:Administration, Oral; D000818:Animals; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007150:Immunohistochemistry; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010634:Phenobarbital / Q000494:pharmacology*; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D051736:Proto-Oncogene Proteins c-mdm2 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D011285:Pregnenolone Carbonitrile; C497208:Mdm2 protein, rat; D051736:Proto-Oncogene Proteins c-mdm2; D010634:Phenobarbital