Abstract
Primary cultures of adult rat hepatocytes grown in serum-free hormonally defined medium were shown, for the first time, to be capable of supporting the 3-methylcholanthrene-inducible expression of cytochrome P-450d. Such cultures were used to investigate the mechanism of the induction of cytochrome P-450c and P-450d mRNAs. After 1 day of growth in culture, P-450c and P-450d mRNAs were induced 33- and 28-fold, respectively, by 3-methylcholanthrene treatment. A similar magnitude of induction was achieved after 2-5 days growth in culture. However, the relative abundance of the two mRNAs before and after treatment decreased linearly over the 5-day time course. Kinetic analysis revealed that induction of both genes was rapid and could be observed less than 2 h following treatment. Accumulation of both mRNAs was linear for 8 h, reaching a plateau by 12 h. Expression then remained constant for at least 12 additional hours. In vitro nuclear run-on experiments revealed a 3.9- and 2.0-fold transcriptional induction of the P-450c and P-450d genes, respectively. This is in contrast to the large induction of accumulation of these mRNAs observed at steady state. Thus, the 3-methylcholanthrene induction of P-450c and P-450d mRNAs in the hepatocyte cultures appeared to be mediated primarily at the post-transcriptional level. Experiments on rat liver showed that, in vivo, P-450d is also regulated primarily at the post-transcriptional level. However, P-450c was found to be regulated primarily transcriptionally.
Highlights
Primary cultures of adult rat hepatocytes grown in The pattern of expression of the two genes in the rat and serum-free hormonally defined medium were shown, inthe mouse indicates that the genes areunder complex for the first time, to be capable of supporting the 3- regulation
The induction mechanism of the P1-450 gene and the orthologous rat P-45Oc gene appears to be mediated by the hydrocarbon-receptor complex in a manner analogous to Kinetic analysis revealed that inductioonf both genes glucocorticoid receptor-mediated transcriptional induction (9, was rapid ancdould be observedless than 2 h following 10)
Cytochrome P-450d mRNA Is Expressed in Primary Hepatocyte Cultures-The absence of a cell culture system capable of supporting 3-methylcholanthrene-induced expression of cytochrome P-450d has hindered a full analysis of the mechanism of its regulation
Summary
1-2 X lo cpm of 32P-labeledRNA was hybridized to 4 pg of purified insert cDNA probe immobilized on nylon membranes as described previously [28]. Filters were washed as described previously, incubated with ribonuclease A (1pg/ml) for 30 min at 37 "C, and rinsed several times with 2 X SSPE at room temperature. Liver nuclei from control and 3-methylcholanthrene-injected rats were prepared as described previously [25]. Of the 416 bases in the fragment, 385 (92%)are identical with the corresponding region of rat P-45Oc sequence [42] with no insertions or deletions in the alignment. The Pa-450 cDNA probe used was a partial cDNA clone of the P3-450 mRNA which includes bases 780-. The first 830 bases have 777 (93%) identical with the rat mRNA with no insertions or deletions in the alignment. Additional cloned DNA fragments used include human y-actin [31], mouse al-antitrypsin (al-AT) [25, 43], and a human skeletal myosin heavy chain 3' end 1.1-kilobasefragment.'
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