Detection of a tyrosine-phosphorylated form of cyclin A during liver regeneration.

Cyclin A functions in both the S and G2-M phases of the cell cycle. The expression of cyclin A during liver regeneration was compared with that of cyclin B1 and p34cdc2. Liver regeneration was followed at 2-h intervals from 12 to 48 h after partial hepatectomy (PH). Immunohistochemical staining using proliferating cell nuclear antigen revealed DNA synthesis peaks at 18 h after PH. The most intense nuclear staining of hepatocytes with cyclins A and B1 and p34cdc2 antibodies occurred at 26 h post-PH, which corresponds with the onset of mitosis. Quantitative mRNA expression of cyclins A and B1 and p34cdc2 was determined by competitive reverse transcription-PCR. Construction of mRNA internal standards and coamplification during reverse transcription-PCR allowed quantitation of all three cell cycle genes. At 24 h post-PH, cyclin A mRNA levels were approximately 5 fg/100 ng total RNA. In contrast, cyclin B1 and p34cdc2 levels were 20-fold higher, 100 fg/100 ng total RNA. Cyclin B1 and p34cdc2 mRNA levels showed two peaks, at 26 and 38-44 h post-PH, whereas the levels of cyclin A were constant during this interval. Immunoblots revealed the presence of cyclin A in normal liver, and significant amounts were present as early as 12 h post-PH. At 26 h post-PH, tyrosine-phosphorylated forms of cyclin A were detected. Cyclin B1 and p34cdc2 protein were not present until 22-24 h post-PH, and two peaks were observed, at 26 and 38-44 h, coinciding with the mRNA pattern. Histone H1 kinase activity was associated with the two peaks of cyclin B1 and p34cdc2 expression. The unique pattern of cyclin A expression and detection of tyrosine-phosphorylated forms suggest a different mechanism for the regulation of cyclin A during liver regeneration.

Effects of c-Jun and a negative dominant mutation of c-Jun on differentiation and gene expression in lens epithelial cells.

We have used a retroviral vector (RCAS) to overexpress wild-type chicken c-Jun or a deletion mutant of chicken c-Jun (Jun delta 7) lacking the DNA binding region to investigate the possible role of c-Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c-Jun increased the rate of cell proliferation and greatly delayed the appearance of "lentoid bodies," structures which contain differentiated cells expressing fiber cell markers. Excess c-Jun expression also significantly decreased the level of beta A3/A1-crystallin mRNA, without affecting alpha A-crystallin mRNA. In contrast, the mutated protein, Jun delta 7, had no effect on proliferation or differentiation but markedly increased the level of alpha A-crystallin mRNA in proliferating cell cultures. These results suggest that c-Jun or Jun-related proteins may be negative regulators of alpha A- and beta A3/A1-crystallin genes in proliferating lens cells.

Expression of c-fos and c-jun mRNA in the developing chicken lens: relationship to cell proliferation, quiescence, and differentiation.

The in vivo developmental pattern of c-fos and c-jun mRNA expression has been examined in the embryonic chicken lens using a coupled reverse transcription/polymerase chain reaction assay. Levels of each mRNA were measured in the central epithelium, equatorial epithelium, and fiber cell mass at 6, 10, 14, and 19 days of development. The results showed that c-fos and c-jun mRNAs accumulated during development of the embryonic chicken lens epithelium as the proportion of proliferating cells decreased, suggesting that quiescent epithelial cells express high levels of both protooncogene mRNAs. Cells in the early stages of terminal differentiation near the lens equator also contained relatively high levels of c-fos and c-jun mRNA. As lens fiber cells matured, the number of copies of c-fos mRNA per cell decreased markedly, while c-jun mRNA increased. These findings demonstrate that c-fos and c-jun are differentially regulated during terminal differentiation of lens fiber cells and suggest that these protooncogenes are expressed in lens epithelial cells following cell cycle arrest.

Kinetics of excretion of 2-acetylaminofluorene in normal and xenobiotic-treated rats and in rats with hepatocyte nodules.

This study was designed to explore further the hypothesis that the special resistance phenotype seen in hepatocyte nodules during liver carcinogenesis could have a physiologic correlate in the manner with which a carcinogenic xenobiotic is handled. Hepatocyte nodules were induced in male rats by continuous or intermittent exposure to dietary 2-acetylaminofluorene over a 25-week period. Two or 5 weeks after the exposure, the animals were given a single dose of 9-14C-2-acetylaminofluorene. The amounts and rates of excretion of unconjugated compound and derivatives and of the glucuronic acid metabolites in the bile and urine and the amounts in the blood and liver were measured over a period of 180 minutes. For comparison, animals fed the basal diet alone, animals injected with phenobarbital or 3-methylcholanthrene, animals receiving a single dose of cobalt heme and animals fed the 2-acetylaminofluorene for only 2 weeks were studied. These groups were used as controls for different patterns of drug metabolism, especially relating to the cytochromes P-450. The nodule-bearing animals showed a pattern of handling of the carcinogen that is quite different than that of the animals of any other group. They excreted in the bile plus urine from 20 to 30% less. However, relatively much more was in the urine. The free and glucuronide-conjugated metabolic products of the carcinogen were assessed by high performance liquid chromatography. The nodule-bearing animals and the animals treated with 3-methylcholanthrene excreted much more glucuronic acid esters. The pattern of distribution of labeled 2-acetylaminofluorene is different in the nodule-bearing rats than in other animals in which variations in phase I and phase II drug-metabolizing enzymes were induced by treatment with cobalt heme, phenobarbital, 3-methylcholanthrene or short-term exposure to dietary 2-acetylaminofluorene.

Kinetics of interaction of 2-acetylaminofluorene with normal liver and carcinogen-induced hepatocyte nodules in vivo and in vitro.

This study examines the initial uptake and subcellular distribution of the carcinogen [14C]-2-acetylaminofluorene in liver nodules and normal liver. The route of administration of the carcinogen was intravenously through a peripheral branch of the superior mesenteric vein, intragastrically or intraperitoneally. Tissue distribution was initially dependent on blood flow, but the retention after 5 minutes varied between different tissues according to tissue affinity, high in liver, fat and muscle, low in kidney and brain. The major fraction was retained in the liver. In vitro experiments demonstrated that total levels of [14C]-2-acetylaminofluorene were 8-fold lower in hepatocytes from liver nodules compared with normal liver. The 2-acetylaminofluorene was bound more avidly to 12 to 15 kilodalton cytosolic proteins than to 40 to 50 kilodalton proteins in normal liver and this binding was much less in hepatocyte nodules. The subcellular distribution indicated that the microsomal fractions had a greater specificity than mitochondria, homogenate, or cytosol. This specificity was not due to the lipid content of the fractions. Microsomal fractions from liver nodules had 2-fold less [14C]-2-acetylaminofluorene bound than from normal liver. The carcinogen was bound in cytosolic proteins with a peak 90 minutes after intravenous injection, as compared with a peak for microsomes at 10 minutes. These results lend further support for the concept that the biochemical properties in liver nodules minimize the metabolism of xenobiotics in vivo.

The pattern of metabolism of 2-acetylaminofluorene in carcinogen-induced hepatocyte nodules in comparison to normal liver.

This study was designed to test further the hypothesis that the special biochemical pattern seen in hepatocyte nodules during liver carcinogenesis could be of fundamental importance in their selective metabolism of one carcinogenic xenobiotic, 2-AAF, as related to their resistance to xenobiotics. Nodules of a certain stage were induced using the resistant hepatocyte model. The metabolism of a single small dose of 2-AAF in hepatocyte nodules in comparison to normal liver was studied at different time intervals up to 30 h. The levels of free 2-AAF in nodules and in normal liver were approximately the same over the whole time period. However, the nodules showed a large decrease in the binding of 2-AAF to DNA, RNA and proteins as well as in the metabolic conversion to hydroxylated forms, both free and conjugated with glucuronic acid. The patterns of metabolic conversion to metabolites and of conjugation of the metabolites are in harmony with the known biochemical patterns in nodules, a decrease in phase I components involved in the metabolism of carcinogens and other xenobiotics and an increase in most phase II components involved in conjugation and detoxification.