cyclin-d1 and 1-4-bis(2-(3-5-dichloropyridyloxy))benzene

Thyroid hormone is known to elicit diverse cellular and metabolic effects in various organs, including mitogenesis in the rat liver. In the present study, experiments were carried out to determine whether thyroid hormone is able to stimulate cell proliferation in another quiescent organ such as the pancreas. 3,5,3'-L-tri-iodothyronine (T3) added to the diet at a concentration of 4 mg/kg caused a striking increase in nuclear bromodeoxyuridine (BrdU) incorporation of rat acinar cells 7 days after treatment (the labeling index was 46.7% in T3-treated rats vs 7.1% in controls). BrdU incorporation was limited to the acinar cells, with duct cells and islet cells being essentially negative. The increase in DNA synthesis was accompanied by the presence of several mitotic figures. Histological examination of the pancreas did not exhibit any sign of T3-induced toxicity. Determination of the apoptotic index, measurement of the serum levels of alpha-amylase and lipase, and glycemia determination did not show any increase over control values, suggesting that the enhanced proliferation of acinar cells was a direct effect induced by T3 and not a regenerative response consequent to acinar or beta-cell injury. Additional experiments showed that DNA synthesis was induced as early as 2 days after T3 treatment (the labeling index was 9.4 vs 1.9% in controls) and was associated with increased protein levels of cyclin D1, cyclin A and proliferating cell nuclear antigen, with no substantial differences in the expression of the cyclin-dependent kinase inhibitor p27. The mitogenic effect of T3 on the pancreas was not limited to the rat, since extensive acinar cell proliferation was also observed in the pancreas of mice treated with T3 for 1 week (the labeling index was 28% in T3-treated mice vs 1.8% in controls). Treatment with three other ligands of nuclear receptors, ciprofibrate, all-trans retinoic acid and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, induced little or no pancreatic cell proliferation. These results demonstrated that T3 is a powerful inducer of cell proliferation in the pancreas and suggested that pancreatic acinar cell proliferation by selected agents may have potential for therapeutic use.

Topics: Administration, Oral; Animals; Biomarkers; Blotting, Western; Bromodeoxyuridine; Cell Proliferation; Clofibric Acid; Cyclin A; Cyclin D1; Female; Fibric Acids; Immunohistochemistry; Male; Mice; Mice, Inbred Strains; Pancreas; Peroxisome Proliferators; Proliferating Cell Nuclear Antigen; Pyridines; Rats; Rats, Inbred F344; Receptors, Cytoplasmic and Nuclear; Stimulation, Chemical; Tretinoin; Triiodothyronine

The nuclear receptor Constitutive Androstane Receptor (CAR) binds DNA as a heterodimer with the retinoic-X receptor and activates gene transcription. Previously, in vitro studies have shown that the testosterone metabolites, androstenol and androstenol, inhibit the constitutive transcriptional activity of CAR, suggesting that differences might exist in the response to CAR-mediated gene activation between different sexes. In this study, we have analyzed the response of female and male CD-1 mice to stimulation of hepatocyte proliferation caused by the CAR ligand TCPOBOP. Results showed that the labelling index of female hepatocytes at 24, 30 and 36 h after treatment was much higher than that found in males. The higher proliferative activity of female hepatocytes was associated with increased hepatic levels of cyclin D1, cyclin A, E2F and enhanced phosphorylation of pRb and p107. The increased mitogenic response of females was associated with higher mRNA levels of CYP2B10, a known target of CAR. Administration of androstenol to TCPOBOP-treated mice caused a reduction of labelling index, which was accompanied by a decrease of CYP2B10 and CAR mRNA levels. In conclusion, the results show that, in addition to microsomal detoxification, another biological response elicited by the CAR ligand TCPOBOP, namely, hepatocyte proliferation, occurs at higher levels in female than male mice, suggesting that CAR transcriptional activity in males is partially counteracted by physiological higher levels of testosterone metabolites such as androstenol and androstenol.

Topics: Androstanols; Animals; Aryl Hydrocarbon Hydroxylases; Cell Division; Constitutive Androstane Receptor; Cyclin D1; Cytochrome P450 Family 2; Female; Hepatocytes; Male; Mice; Pyridines; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Sex Characteristics; Steroid Hydroxylases; Transcription Factors

Cyclin D1 is considered to play a critical role in the progression from G1 to S phase of the cell cycle, and its overexpression is seen in many human tumors. However, previous studies in cell lines have shown that cyclin D1 is not sufficient to trigger cell replication. To directly test the role of cyclin D1 in the progression of the cell cycle, we have examined the proliferative response of hepatocytes to the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mice with homozygous disruption of the cyclin D1 gene. We found that 24 hours after administration of TCPOBOP, the number of bromodeoxyuridine (BrdU)-positive hepatocytes was significantly reduced in cyclin D1(-/-) (labeling index was 1.9% in knockout mice vs. 9.7% of heterozygous mice); however, no difference in the number of proliferating hepatocytes was found 36 or 72 hours after treatment (labeling index was 16% and 43% in cyclin D1(-/-) mice vs. 20% and 41% of heterozygous mice), indicating that lack of cyclin D1 may transiently delay entry into S phase but is not sufficient to inhibit the response of hepatocytes to mitogenic stimuli. The results also show that although there was no difference in hepatic protein levels of cyclin D2 and D3 between untreated cyclin D1(-/-) and cyclin D1(+/-) mice, messenger RNA (mRNA) and protein levels of cyclin E were much higher in the former. In conclusion, our results show that cyclin D1 is not essential for liver development and hepatocyte proliferation induced by mitogenic stimuli and suggest that overexpression of cyclin E may compensate for the lack of cyclin D1.

Topics: Animals; Blotting, Western; Cell Division; Cyclin D1; Cyclin E; DNA; Gene Expression; Hepatocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogens; Pyridines; RNA, Messenger; S Phase

We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.

Topics: Animals; Bromodeoxyuridine; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Gene Expression; Genes, p53; Hepatectomy; Liver; Mice; Mice, Inbred Strains; Microtubule-Associated Proteins; Mitogens; Pyridines; S Phase; Time Factors; Tumor Suppressor Proteins