Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation.

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.

Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 alpha-subunit genes.

The alpha-subunit of the casein protein kinase CK2 has been implicated in both light-regulated and circadian rhythm-controlled plant gene expression, including control of the flowering time. Two putative CK2alpha genes of perennial ryegrass (Lolium perenne L.) have been obtained from a cDNA library constructed with mRNA isolated from cold-acclimated crown tissue. The genomic organisation of the two genes was determined by Southern hybridisation analysis. Primer designs to the Lpck2a-1 and Lpck2a-2 cDNA sequences permitted the amplification of genomic products containing large intron sequences. Amplicon sequence analysis detected single nucleotide polymorphisms (SNPs) within the p150/112 reference mapping population. Validated SNPs, within diagnostic restriction enzyme sites, were used to design cleaved amplified polymorphic sequence (CAPS) assays. The Lpck2a-1 CAPS marker was assigned to perennial ryegrass linkage group (LG) 4 and the Lpck2a-2 CAPS marker was assigned to LG2. The location of the Lpck2a-1 gene locus supports the previous conclusion of conserved synteny between perennial ryegrass LG4, the Triticeae homoeologous group 5L chromosomes and the corresponding segment of rice chromosome 3. Allelic variation at the Lpck2a-1 and Lpck2a-2 gene loci was correlated with phenotypic variation for heading date and winter survival, respectively. SNP polymorphism may be used for the further study of the role of CK2alpha genes in the initiation of reproductive development and winter hardiness in grasses.

Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3).

The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

Ciprofibrate and triiodothyronine do not suppress in vivo induction of placental glutathione S-transferase expression in rat hepatocytes.

Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.

Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas.

Previous studies have demonstrated that short-term treatment with peroxisome proliferators decreased the size and number of gamma-glutamyl transpeptidase or placental glutathione S-transferase (GSTP)-positive hepatic hyperplastic lesions. In this study, we have examined the effect of the hormone triiodothyronine (T3), which, similarly to peroxisome proliferators, is a strong liver mitogen and a ligand of nuclear receptors, on the growth of GSTP-positive nodules generated by the resistant hepatocyte model and on the development of hepatocellular carcinoma. Hepatic hyperplastic nodules were induced in male Fischer rats by a single dose (150 mg/kg) of diethylnitrosamine, followed by a 2-week exposure of the animals to 2-acetylaminofluorene and partial hepatectomy. Nine weeks after diethylnitrosamine administration, rats were switched to a diet containing 4 mg/kg T3 for 1 week (experiment 1) and sacrificed during T3 feeding or were exposed to seven cycles of T3-supplemented diet (1 week/month per 7 months), and sacrificed 6 months after the last cycle (experiment 2). Results showed that T3 treatment for 1 week caused a 70% reduction in the number of GSTP-positive nodules (14/cm2 in T3-fed rats versus 44/cm2 of control animals), as well as GSTP-positive area (12% versus 43% of controls). Reduction in the number of GSTP-positive nodules observed 1 week after T3 feeding was associated with a strong increase in the labeling index of enzyme-altered nodules compared with that of controls (labeling index was 64 and 31%, respectively). No significant differences in the apoptotic index were observed between the two groups. Results from experiment 2 did reveal that although rats treated with diethylnitrosamine + 2-acetylaminofluorene developed 100% hepatocellular carcinoma and 33% of them showed lung metastasis, only 50% of rats exposed to repeated cycles of triiodothyronine developed hepatocellular carcinoma with no lung metastasis. This study indicates that cell proliferation per se might not necessarily represent a promoting condition for putative preneoplastic lesions and demonstrates an anticarcinogenic effect of T3.

Early increase in cyclin-D1 expression and accelerated entry of mouse hepatocytes into S phase after administration of the mitogen 1, 4-Bis[2-(3,5-Dichloropyridyloxy)] benzene.

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.

Cell proliferation induced by 3,3',5-triiodo-L-thyronine is associated with a reduction in the number of preneoplastic hepatic lesions.

Previous studies have suggested that liver cell proliferation is fundamental for the growth of carcinogen-initiated cells. To gain further information on the association between cell proliferation and hepatocarcinogenesis, we have examined the effect of the hormone 3,3',5-triiodo-L-thyronine (T3), a strong liver mitogen, on the growth of diethylnitrosamine (DENA)-induced hepatic lesions positive for the placental form of glutathione S-transferase (GSTP). Two weeks after a single initiating dose of DENA (150 mg/kg), cycles of liver cell proliferation were induced in male Fischer rats by feeding a T3-supplemented diet (4 mg/kg) 1 week/month for 7 months. Rats were killed at the end of the seventh cycle or 1 month later. Results indicate that, in spite of an increased labelling index, a 70% reduction in the number/cm(2) of GSTP-positive minifoci occurred in T3-treated rats. A decrease in the number of GSTP-positive foci was also observed in T3-treated rats killed 1 month after the last exposure to the hormone (40, versus 67 foci/cm(2) in controls), indicating that the reduction was not due to an inhibitory effect on GSTP exerted by the concomitant presence of T3. In a second series of experiments where DENA-treated rats were fed T3 for 1 week and then subjected to the resistant hepatocyte (RH) model, it was found that T3 treatment prior to promotion resulted in a decrease in the number of GSTP-positive foci (16 GSTP(+) foci/cm(2) in T3-fed animals versus 45 in the control group). The results indicate that cell proliferation associated with T3 treatment: (i) reduces the number of carcinogen-induced GSTP-positive lesions; (ii) does not exert any differential effect on the growth of the remaining foci; (iii) inhibits the capacity of putative DENA-initiated cells to be promoted by the RH model. Data suggest that cell proliferation may not necessarily represent a stimulus for the growth of putative preneoplastic lesions.

Hepatic hyperplasia and cancer in rats: alterations in copper metabolism.

We previously demonstrated that rats exposed to the peroxisome proliferator (PP) diethylhexylphthalate (DEHP) had reduced serum ceruloplasmin (CP) oxidase activity, which suggests tissue copper deposition. Copper is highly toxic in excess, and results in cellular damage and hepatocellular carcinomas (HCC). This study addresses changes in expression of copper-related genes and metal accumulation in hyperplastic liver and tumors induced by PP. Male rats were fed diets containing DEHP or clofibrate (CLF) for 3-60 days (hyperplasia) and 4-chloro-6-(2,3 xylidino)-2-pyrimidinyl-thio(N-beta-hydroxyethyl) acetamide for 10 months (HCC). During hyperplasia, an immediate and progressive decrease in serum CP activity was observed (P < 0.05), as were reductions in mRNA levels for both CP and Wilson's disease gene (WD gene, a P-type ATPase) (P < 0.05). Tumor-bearing rats had lower serum CP activity (P < 0.05), and CP and WD gene mRNA levels were reduced in tumors (P < 0.05), and in liver surrounding tumors (SL) (P < 0.05). Metallothionein mRNA showed no consistent changes during hyperplasia. Tumors showed a 2.5-fold induction of metallothionein mRNA (P < 0.05), and a 1.2-fold increase in SL. Temporal increases in liver copper content occurred during hyperplasia, with increases of 2-fold (DEHP) and 3.3-fold (CLF) at 60 days (P < 0.05). Copper content was 2.2-fold higher in tumors (P < 0.05) and 1.7-fold higher in SL; iron did not increase and zinc decreased temporally. Thus, copper accumulation and changes in copper-related gene expression may be contributing factors in liver neoplasia in PP-treated rats. Loss of CP results in decreased free radical scavenger capacity and thus may enhance oxidative damage induced by PPs.

In vivo hepatocyte proliferation is inducible through a TNF and IL-6-independent pathway.

Recent studies in mice harboring a targeted disruption of genes encoding TNF receptor 1 (TNFR-1) or Interleukin 6 (IL-6) suggested a critical role for TNF and IL-6 in initiation of liver regeneration after 2/3 partial hepatectomy. However, hepatocyte proliferation can also occur following treatment with agents that do not induce tissue loss (primary mitogens). To determine whether the above cytokines could also be involved in mitogen-induced liver cell proliferation, we studied the hepatocyte proliferative response after treatment with primary mitogens in mice knock-out for TNFR-1 or IL-6. Our results showed no difference in the proliferative response of the liver between the wild type and the knock-out mice following treatment with the mitogens 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or the peroxisome proliferator, ciprofibrate, suggesting that TNF or IL-6 may not play a major role in this type of proliferation. Gel shift assay indicated that TCPOBOP-induced hepatocyte proliferation is not associated with activation of STAT3 transcription factor, a major target of IL-6 and other growth factors/cytokines. Our results thus indicate that hepatocyte proliferation can be induced by at least two different pathways; compensatory regeneration being TNF and IL-6-dependent, and mitogen-induced direct hyperplasia which does not require TNF or IL-6.

Glucocorticoid-induced effects on pattern formation and epithelial cell differentiation in early embryonic rat lungs.

In this study, we examined the effects of dexamethasone (DEX) on airway branching and subsequent lung maturation. DEX treatment of fetal rat lung explants was initiated during the early pseudoglandular stage of development. Day 14 fetal lung explants were cultured with and without DEX for 4 d. Explants treated with 10 nM or higher concentrations of DEX showed features of both distorted and accelerated maturation. DEX-treated lungs had growth retardation, distorted branching, dilated proximal tubules, and suppressed proliferation of epithelial cells of the distal tubules. Several biochemical and morphologic features of accelerated maturation were also observed: 1) the epithelial cells lining the distal tubules (prospective respiratory airways) were generally cuboidal or flattened; 2) the cuboidal cells often contained lamellar bodies and abundant glycogen; 3) rudimentary septa and large airspace were present; 4) mesenchymal tissue was attenuated and compressed between adjacent epithelial tubules; 5) the distribution of SP-C mRNA in distal tubules was more mature, with individual and clusters of cells expressing SP-C transcripts; and 6) the transcript levels of several genes related to epithelial growth [keratinocyte growth factor (KGF), KGF receptor, and hepatocyte growth factor receptor] and differentiation [surfactant proteins, SP-A, SP-B and SP-C and the Clara cell secretory protein, CC10] were precociously increased. These results show that DEX treatment of the lung during the early pseudoglandular stage accelerates the acquisition of several features of advanced maturation that normally accompany late stages of fetal development. We postulate that KGF mediates at least some effects of DEX on lung maturation and gene expression.

Antagonistic effects of dexamethasone and retinoic acid on rat lung morphogenesis.

We have reported that dexamethasone (DEX) treatment of early embryonic rat lungs in culture induced features of both distorted and accelerated maturation. In this report, we investigated the effects of retinoids on normal and DEX-induced lung development in vitro. Lung maturation was assessed by examining the morphology and the expression of genes related to epithelial differentiation (surfactant proteins, SP-A, SP-B and SP-C and Clara cell protein, CC10) and growth [keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF)]. We cultured d 14 and 15 fetal rat lungs in the presence of DEX (1-1000 nM) and/or all-trans-retinoic acid (RA) (10(-7)-10(-5) M) for 4 d. RA at 10(-6) and 10(-5) M inhibited branching and dilated the distal tubules, and at 10(-5) M caused dilatation of the proximal tubules destined to form the trachea and the main bronchi. The adverse effects of DEX, such as distorted branching, tubular dilatation, and suppression of both lung growth and epithelial cell proliferation, were all prevented by RA. In addition, RA inhibited several features of DEX-induced accelerated maturation, such as: 1) the increased levels of SP-A, SP-B, and CC10 mRNAs; 2) the attenuation of mesenchymal tissue; and 3) the mature distribution of cells expressing SP-C mRNA. In contrast, RA potentiated the increase of KGF and decrease of HGF transcripts induced by DEX. In conclusion, the study shows antagonism by RA of DEX-induced effects on lung morphology and gene expression. We postulate that normal lung development requires a balanced action of endogenous retinoids and glucocorticoids.

Enhancement of aflatoxin B1-induced enzyme altered hepatic foci in rats by treatment with carbon tetrachloride.

The effect of carbon tetrachloride (CCl4) on aflatoxin B1 (AFB1)-induced enzyme altered hepatic foci has been examined in young male Fischer rats given AIN-76A diet. A single i.p. dose of AFB1 (0.2 mg/kg body wt) was given to rats 24 h after partial hepatectomy. Two weeks later, CCl4 (0.8 ml/kg body wt) was injected i.p. once a week for 9 weeks. Animals were sacrificed 24 h after the last dose of CCl4 and glutathione S-transferase placental form (GST-P) and gamma-glutamyl transpeptidase (GGT) positive hepatic foci were analyzed by immunohistochemical and histochemical methods, respectively. Ten weeks after AFB1 dosing, treatment with CCl4 increased the number of AFB1-induced enzyme altered foci several fold and produced a ten to twenty-fold increase in area and volume. GST-P was more sensitive than GGT in detecting AFB1-induced enzyme altered foci. Treatment with AFB1 or CCl4 produced mild hepatic fibrosis in zones 1 and 3 respectively, whereas both treatments produced severe fibrosis in zones 1 to 3 areas. Treatment with CCl4 after AFB1 dosing lowered hepatic GSH levels by 20% and increased lipid peroxidation by 40%. It appears that CCl4, by being an effective enhancer of AFB1-induced enzyme altered hepatic foci in the rat, may mimic cirrhosis observed in human hepatocellular carcinoma.

Hepatic hyperplasia and cancer in rats: metabolic alterations associated with cell growth.

BACKGROUND & AIMS: We showed previously that the peroxisome proliferators di(2-ethylhexyl)phthalate (DEHP), clofibrate, and 4-chloro-6-(2,3 xylidino)-2-pyrimidinylthio (N-beta-hydroxyl)acetamide (BR931) alter hepatic sex steroid metabolism and receptor expression during induction of hepatic hyperplasia and hepatocellular carcinoma (HCC) in rats. The aim of this study was to identify metabolic changes associated with cell growth during hyperplasia and HCC. METHODS: Hepatic hyperplasia was induced in male rats by a diet containing DEHP and clofibrate for 3-60 days. HCC was induced by feeding a diet containing BR931, a more potent hepatocarcinogen, for 10 months. RESULTS: Cholesterol biosynthesis was depressed in hyperplastic livers but increased in HCC. Glucose-6-phosphate dehydrogenase (G6PD) activity was inhibited in hyperplastic liver as well as in HCC, whereas malic enzyme activity increased severalfold. Protein and messenger RNA (mRNA) levels for both G6PD and malic enzyme increased in hyperplastic livers and HCC. mRNA levels for 3-hydroxy-3-methylglutaryl-coenzyme A reductase decreased in hyperplasia and increased in HCC, whereas low-density lipoprotein receptor mRNA increased in hyperplasia and decreased in HCC. CONCLUSIONS: Neoplastic cells acquire a growth advantage by their capacity to synthesize cholesterol and obtain reduced nicotinamide adenine dinucleotide phosphate by the malic enzyme pathway when G6PD activity is inhibited by peroxisome proliferators.

Induction of cellular DNA synthesis in the pancreas and kidneys of rats by peroxisome proliferators, 9-cis retinoic acid, and 3,3',5-triiodo-L-thyronine.

We recently suggested that peroxisome proliferators (PPs), 3,3',5-triiodo-L-thyronine (T3), and 9-cis retinoic acid (9-cis RA) induce hepatocyte proliferation in rats through the activation of their nuclear receptors, PP-activated receptors, T3 receptors, and retinoid X receptors. To test whether nuclear hormone receptor-mediated cell proliferation can be observed in organs other than liver, we examined the effects of these agents on the pancreas and kidneys of male Wistar rats using BrdUrd immunohistochemistry. A single s.c. injection of T3 (2 mg/kg) and single intragastric administration of 9-cis RA (40 mg/kg) or 4-chloro-6-(2, 3-xylidino)-2-pyrimidinylthio-(N-beta-hydroxyethyl) acetamide (200 mg/kg) induced a wave of DNA synthesis in the pancreatic acinar cells and in the proximal tubular epithelial cells of the kidneys, peaking after 24 h. No stimulation of DNA synthesis was observed in ductal or islet cells of the pancreas and in glomeruli of the kidneys. All-trans-retinoic acid, a ligand for retinoic acid receptor, at a dose (200 mg/kg) that induced hepatocyte proliferation, had no effects on cell proliferation of the pancreas and the kidneys. The results suggest that T3, 9-cis RA, and PP activate genes that regulate cell proliferation in target cells through receptor-mediated pathways and initiate cellular DNA synthesis.

Liver cell proliferation induced by nafenopin and cyproterone acetate is not associated with increases in activation of transcription factors NF-kappaB and AP-1 or with expression of tumor necrosis factor alpha.

Our previous studies have shown a different pattern of immediate early gene and growth factor gene expression between compensatory liver regeneration occurring after cell loss/death and direct hyperplasia induced by primary mitogens. In the present study, modifications in the activation of two transcription factors, NF-kappaB and AP-1; steady-state levels of tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA); and induction of the inducible nitric oxide synthase (iNOS) were examined in rat liver during different types of cell proliferation. Compensatory regeneration was induced in male Wistar rats by partial hepatectomy of two thirds (PH) or a necrogenic dose of CCl4 (2 mL/kg), whereas direct hyperplasia was induced by a single administration of the primary mitogens lead nitrate (LN, 100 micromol/kg), cyproterone acetate (CPA, 60 mg/kg), or nafenopin (NAF, 200 mg/kg). Liver regeneration after treatment with CCl4 was associated with an increase in steady-state levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, and induction of iNOS. A strong and prolonged activation of NF-kappaB but not of AP-1 was observed in LN-induced hyperplasia. LN also induced an increase in hepatic levels of TNF-alpha and iNOS mRNA. On the other hand, direct hyperplasia induced by two other primary mitogens, NAF and CPA, occurred in the complete absence of modifications in the hepatic levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, or induction of iNOS, although the number of hepatocytes entering S phase 18 to 24 hours after NAF was similar to that seen after PH. These results add further support to the hypothesis that cell proliferation occurring in the absence of cell loss/death may be triggered by unknown signaling pathways different from those responsible for the transition of hepatocytes from G0 to G1 after PH or cell necrosis.

Increased expression of c-fos, c-jun and LRF-1 is not required for in vivo priming of hepatocytes by the mitogen TCPOBOP.

The notion that an increased expression of immediate early genes such as c-fos and c-jun is an absolute requirement for the G0-G1 transition of the hepatocytes has recently been challenged by the finding that rat liver cell proliferation induced by primary mitogens may occur in the absence of such changes (Columbano and Shinozuka, 1996). To further investigate the relationship between immediate early genes and hepatocyte proliferation, we have compared the hepatic levels of c-fos, c-jun and LRF-1 transcripts during mouse liver cell proliferation in two conditions: (i) direct hyperplasia induced by the non-genotoxic hepatocarcinogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and (ii) compensatory regeneration caused by a necrogenic dose of carbon tetrachloride. The results show striking differences in the activation of early genes. In spite of a rapid stimulation of S phase by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (approximately 8% of hepatocytes were BrdU-positive as early as 24 h after mitogen treatment versus 1% of labelled hepatocytes after 2/3 partial hepatectomy), no changes in the expression of c-fos, c-jun and LRF-1 could be observed. Moreover, no change in steady state mRNA hepatic levels of IGFBP-1 (a gene highly expressed in rat liver following partial hepatectomy), and only a slight increase in c-myc and PRL-1, was found after mitogen administration. On the contrary, a rapid, massive and transient increase in the hepatic mRNA levels of all these genes was observed during carbon tetrachloride induced regeneration. The results indicate that increased expression of immediate early genes may be dependent upon the nature of the proliferative stimulus, and it may not be a prerequisite in certain in vivo conditions such as proliferation induced in the absence of liver tissue damage.

Potentiation of aflatoxin B1-induced hepatocarcinogenesis in the rat by pretreatment with buthionine sulfoximine.

A single i.p. dose of aflatoxin B1 (AFB1) (1.0 and 2.0 mg/kg body wt)-induced hepatocarcinogenesis with phenobarbital as a promoter has been examined in young male Fischer rats. Immunohistochemical method has been employed to detect AFB1-induced glutathione S-transferase placental form (GST-P)-positive hepatic foci observed from 3 week and 10 week to 40-48 week periods. With 2.0 mg AFB1 dosing, the number, area and volume occupied by GST-P-positive hepatic foci increased significantly and progressively from 3 week, 10 week and 48 week periods. In long term studies (40-48 weeks), 1.0 mg and 2.0 mg AFB1 dose levels yielded linear response in area and volume occupied by AFB1-induced hepatic foci. Pretreatment of rats with L-buthionine sulfoximine (BSO), a GSH depleter, at a dose of 4 mmol/kg body wt 4 and 2 h before 1.0 or 2.0 mg AFB1 treatment enhanced the number, area and volume of GST-P-positive hepatic foci, increases being the largest at shorter time periods (3 and 10 weeks) compared to longer time periods (40 and 48 weeks). This report appears to be the first example of an enhanced chemical induced hepatocarcinogenesis in a long term study in any experimental animals species by a GSH depleting agent.

Effect of cell proliferation on initiation of aflatoxin B1-induced enzyme altered hepatic foci in rats and hamsters.

Rat is susceptible whereas hamster is resistant to aflatoxin B1 (AFB1) hepatocarcinogenesis. Effect of cell proliferation on AFB1-induced glutathione S-transferase placental form (GST-P) positive foci has been examined in these two species after a single i.p. dose of AFB1 and phenobarbital (PB) as a promoter in a 3 week period. Bromodeoxyuridine incorporation as a measure of cell proliferation and GST-P hepatic foci were analyzed by immunohistochemical methods. Hepatic cell proliferation was maximum at 24 h after either partial hepatectomy (PH) or CCl4 (4 mmol/kg) pretreatment of rats whereas cell proliferation was maximum at 48 h after PH or CCl4 (1 mmol/kg) treatment of hamsters. Enhanced number of GST-P positive hepatic minifoci (two to nine cells) and foci (>100 microns) and focal area were observed in rats with either AFB1 (0.5 mg/kg) given 24 h after PH or AFB1 (0.5 or 2.5 mg/kg) given 48 h after CCl4 dosing. In hamsters, 1 or 2 mg AFB1 treatment produced only GST-P positive single hepatocytes without presence of any minifoci whereas 3 or 6 mg AFB1 produced minifoci consisting only of doublets. Pretreatment with CCl4 48 or 72 h before 1 mg AFB1 dose level increased GST-P positive single cells and minifoci several fold. PH 24 or 48 h before 1 or 2 mg AFB1 dose level increased minifoci. However, increase in minifoci was higher in PH hamsters at 48 h compared with those at 24 h. These results indicate that even though maximum initiation occurs in both speices when AFB1 is administered at the peak of DNA synthesis, rats are more responsive than hamsters to cellular proliferation in the initiation phase of AFB1-induced hepatocarcinogenesis.

Keratinocyte growth factor and embryonic rat lung morphogenesis.

We examined possible roles of keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) in lung morphogenesis. By polymerase chain reaction, transcripts for both KGF and its receptor were detected early (rat gestational days 16 and 14, respectively) and their abundance increased during lung morphogenesis. To evaluate possible role of KGF in lung morphogenesis, day 14 lung explants were cultured in Dulbecco's modified Eagle medium + 10% fetal calf serum for 1 to 4 days in the presence (5-50 ng/ml) or absence of KGF (control). KGF (at 25 and 50 ng/ml) induced a marked reduction in the number of terminal branches and destination of the distal epithelium into cyst-like structures. These effects of exogenous KGF were progressively diminished by increasing concentrations of anti-KGF (2-16 micrograms/ml). Electron microscopic examination revealed that the epithelial cells of the cystic structures contained lamellar bodies, and were therefore type II cells and/or their progenitors. Northern blot analysis showed higher expression of surfactant protein C (SP-C) mRNA (a marker for alveolar epithelial type II cells) in KGF-treated fetal lungs. In situ hybridization of the KGF-treated lungs revealed that the SP-C mRNA-expressing cells were arranged distally in the form of linear arrays, a pattern distinctly different from that in control lungs. Acidic fibroblast growth factor, which also binds KGF receptors, in the presence of heparin mimicked the effect of KGF on branching. Transforming growth factor-beta(1) (TGF-beta 1) inhibited branching of fetal lungs in culture, and this effect dominated over that induced by KGF. Blocking of endogenous HGF with antibodies or addition of HGF to cultures of fetal lung explants had no significant effect on branching or growth. In conclusion, KGF markedly influences branching, and epithelial growth, differentiation, and patterning during lung morphogenesis.

Liver regeneration versus direct hyperplasia.

Liver cell growth can be induced in two distinct patterns: compensatory regeneration and direct hyperplasia. In the former, DNA synthesis is preceded by a loss of liver cells such as seen after partial resection of the liver or cell necrosis, whereas in direct hyperplasia, DNA synthesis is stimulated without cell loss. During the past decade, considerable advances have been made in understanding molecular mechanisms of the compensatory regeneration. There is increasing evidence that hepatocyte proliferation induced by some primary mitogens is mediated by patterns of growth factor modulation and signal transduction different from those of compensatory regeneration. Indeed, whereas activation of transcription factors such as NF-kappa B and increased expression of immediate early genes such as c-fos, c-jun, egr-1, and c-myc are induced during compensatory regeneration, such changes are not observed during hyperplasia induced by certain primary mitogens. In addition, although experimental evidence suggests a critical role for growth factors such as hepatocyte growth factor and transforming growth factor-alpha for the progression into cell cycle of competent hepatocytes in compensatory regeneration, these growth factors do not appear to play a major role in direct hyperplasia. One class of primary mitogens may trigger their actions through tumor necrosis factor-alpha, and the other by activation of nuclear hormone receptors. The differences in molecular events observed between liver regeneration and direct hyperplasia may affect differently the initiation step of chemical hepatocarcinogenesis. Whereas the former supports initiation by chemicals, the latter does not. A similar lack of effect on promotion of carcinogen-altered cells has also been observed after acute treatment with some primary mitogens. Definition of the mechanisms by which primary mitogens stimulate liver cell proliferation may elucidate the nature of the signals responsible for triggering the entry into cell cycle. Furthermore, due to their low toxicity, primary liver mitogens could have significant clinical applications in gene transfer and liver transplantation.