Hisashi Shinozuka

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.

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.

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.

Choline Deficiency and Chemical Carcinogenesis

We have reviewed the current status of our knowledge concerning the biologic effects of dietary choline (lipotrope) deficiency in modifying chemical carcinogenesis in experimental animals and discussed its possible mechanisms. Choline deficiency produces various pathologic lesions, involving virtually every organ of the body, as a result of a decrease in phospholipid and acetylcholine synthesis and in the supply of labile methyl groups. The liver is the only organ in which a relationship has been consistently demonstrated between choline deficiency and chemically induced tumors. The deficient diet enhances the initiating potency of several carcinogens and acts as a strong cocarcinogen. Diet also exerts a strong promoting effect, though the possibility that it is a complete carcinogen cannot be ruled out. Phase I enzymes of the carcinogen metabolizing system are uniformly depressed by choline deficiency, but very little information is available regarding the effects of diet on Phase II enzymes that detoxify carcinogen metabolites. Possible modifications of carcinogen-induced DNA damage and their repair processes have not been adequately scrutinized. Solid evidence suggests that feeding a choline-deficient diet leads to enhanced liver cell proliferation, an inadequate supply of methyl groups for transmethylation reactions, and membrane lipid peroxidation. Induced cell proliferation and hypomethylation of DNA may alter the state of gene expression, including that of specific cellular oncogenes. Lipid peroxidation may alter the structure and function of membrane receptors related to liver cell growth or may directly damage cellular DNA. Thus these alterations, individually or in combination, could play a critical role in the diet-induced modification of chemical carcinogenesis.

Free Radical Injury and Liver Tumor Promotion

One of the underlying mechanisms of tumor promotion both in the skin and liver involves free radical mediated injury to informational macromolecules of target cells. A choline-deficient (CD) diet, which is an efficient liver tumor promoter, induces peroxidative damage of liver cell membrane lipids. By modifying components of a CD diet, we have shown that the efficacy of the promotion is correlated with the extent of lipid peroxidation. The substitution of fats in a CD diet with predominantly polyunsaturated fat and the addition of methapyrilene to a CD diet enhances membrane lipid peroxidation and the promoting effects. An antioxidant (BHT) and hypolipidemic peroxisome proliferators (BR.931 and DEHP) suppress both of these effects. Contrary to these findings, phenobarbital did not induce membrane lipid peroxidation, and its addition to a CD diet inhibited the diet-induced lipid peroxidation, though such a combination exerted a stronger promoting action. Thus, a CD diet and phenobarbital exert their promoting actions through different mechanisms. The consequence of membrane lipid peroxidation in the liver cells induced by a CD diet may be multiple. Our recent study of surface membrane insulin receptors of liver cells of rats fed a CD diet showed a decrease in number and an enhanced binding affinity leading to altered responsiveness of liver cells to insulin mediated glycogen synthesis. It is suggested that CD diet-induced lipid peroxidation leads to functional alterations of membrane receptors involved in cell growth control and may thereby exert its promoting action.

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.

Effects of a Choline-deficient Diet and a Hypolipidemic Agent on Single Glutathione S-Transferase Placental Form-positive Hepatocytes in Rat Liver

Using the placental form of glutathione S‐transferase (GST‐P) as a marker of carcinogen‐initiated hepatocytes, we investigated how a choline‐deficient (CD) diet and BR931, a carcinogenic hypolipidemic agent, modify populations of single GST‐P‐positive hepatocytes. The liver of male Fischer rats (6‐7 weeks old) fed a CS or basal diet contained mostly single or double GST‐P‐positive hepatocytes. Feeding a CD diet for 2–4 weeks led to increases in the number of aggregates of two and three GST‐P‐positive hepatocytes. By 8–12 weeks, there was an emergence of discrete foci of GST‐P‐positive hepatocytes consisting of more than 20 hepatocytes. Feeding a BR931 diet for 4–8 weeks resulted in no significant change in the number of single GST‐P‐positive hepatocytes in the liver as compared to feeding a basal diet. It is suggested that single GST‐P‐positive hepatocytes in the liver of relatively young rats maintained on a commercial diet may represent endogenously initiated cells. A CD diet promotes endogenously initiated cells to form larger aggregates or foci of GST‐P‐positive cells.

Elevated levels of prostaglandin E2 in the liver of rats fed a choline deficient diet: possible involvement in liver tumor promotion

The effect of feeding a choline deficient (CD) diet, an efficient liver tumor promoting regimen, on the prostaglandin metabolism in the liver of male Sprague-Dawley rats was investigated. The possible biological significance of the alteration was examined using hypolipidemic peroxisome proliferators and modifiers of prostaglandin metabolism such as indomethacin and menhaden oil in the short term assay of the induction of enzyme altered foci in the liver. A CD diet, when fed for 10-30 days, induced 2-2.5 times increases in the levels of prostaglandin E2 (PGE2) in the liver, while the hypolipidemic peroxisome proliferators, 4-chloro-6(2,3-xylidino) pyrimidinylthio(N-hydroxyethyl)-acetamide (BR931) and di(2-ethylhexyl)-phthalate (DEHP), markedly reduced the levels of this metabolite. The addition of BR931 or indomethacin to a CD diet suppressed the diet-induced elevations of PGE2 and a substitution of fats in a CD diet with menhaden oil had the same effect. Furthermore, both indomethacin and menhaden oil added to a CD diet suppressed the induction of gamma-glutamyltranspeptidase positive hepatocyte foci in the liver of rats initiated with a single dose of diethylnitrosamine after 8 weeks of the dietary promotion. The results suggest that altered prostaglandin metabolism may be involved in the liver tumor promoting effect of a CD diet.

9-cis retinoic acid is a direct hepatocyte mitogen in rats

We recently suggested that peroxisome proliferators (PP)-induced hepatocyte DNA synthesis may be mediated by a specific peroxisome proliferator activated receptor (PPAR). Heterodimers of the PPAR with the retinoid nuclear receptor, RXR, activate transcription after binding to DR1 response elements of the target genes. DR1 elements are also activated by RXR homodimers formed in the presence of 9-cis retinoic acid (9 cis RA) suggesting that PP and 9 cis RA might regulate an overlapping set of target genes. The present study was therefore designed to test whether 9-cis RA stimulates hepatocyte DNA synthesis. Male Wistar rats were given a single intragastric dose of 9-cis RA (10-100 mg/Kg) or all trans retinoic acid (RA)(200 mg/Kg and 100 mg/Kg), and levels of hepatocyte DNA synthesis after 24 hours were determined by BrdU immunohistochemistry. Effects of 9-cis RA and RA(10(-9)-10(-5)M) on hepatocyte DNA synthesis in primary culture were also examined. Over 10 fold increases in the levels of BrdU incorporation were noted 24 hours after a single dose of 9 cis RA at a dose of 60 and 100 mg/Kg. RA at a dose of 200 mg/Kg induced a 5-6 fold increases in BrdU labeling, while a dose of 100 mg/Kg had no significant effects. Since the RA effect only occurs at higher doses, it may be only after conversion to 9-cis RA. In primary culture of hepatocytes, neither 9-cis RA nor RA with or without EGF had stimulatory effects on hepatocyte DNA synthesis. This is the first report to demonstrate a potent stimulatory effect of 9-cis RA on DNA synthesis of rat hepatocytes in vivo. It is suggested that 9-cis RA exerts this effect through receptor mediated mechanisms similar to PP, both activating genes that regulate hepatocyte proliferation.

Multiple atypical acinar cell nodules of the pancreas

Although acinar cell nodules of the pancreas have been described in rats given carcinogenic chemicals, similar nodules have not been reported in humans. This report describes two cases of nodular acinar cell lesions in the pancreas in patients who died of insulin secreting islet cell adenoma and of bronchogenic carcinoma. The lesions consisted of multiple nodules that were well demarcated from the surrounding acinar tissue and were composed of zymogen granule containing acinar cells with a pale to pink cytoplasm. The significance of these atypical acinar cell nodules in regard to their being possible precursor lesions of acinar cell carcinoma of the pancreas is discussed.