Reformation of nucleoli after ethione-induced fragmentation in the absence of significant protein synthesis.

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90-95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed.

The disorganization of hepatic cell nucleoli induced by ethionine and its reversal by adenine.

The structure of nuclei and nucleoli of hepatic cells after short-term ethionine administration was investigated with the electron microscope. By 1(1/2) hr after the injection, a distinct alteration occurred in the nucleoli which was characterized by the appearance of electron-opaque masses in the nucleolonema. After 6-8 hr, the nucleoli showed partial fragmentation into small, dense masses. Large aggregates of interchromatinic granules appeared in the nucleoplasm. Condensation of chromatin became prominent in the nucleoplasm particularly along the nuclear membrane. By 12 hr almost complete fragmentation of nucleoli had occurred. The administration of adenine or methionine at 4 hr prevented the development of nucleolar changes. Also, adenine administration at 8 hr after ethionine completely reversed the nucleolar lesion by 12 hr. After methionine administration at 8 hr, many nucleoli showed incomplete reconstruction with many twisted ropelike structures when viewed 4 hr later. Identical structures were found when adenine was given at 8 hr, and animals were sacrificed 2 hr later. On the basis of this observation, the simplified structures of nucleoli found 2 hr after adenine or 4 hr after methionine appeared to be precursors of the nucleolonema. It is suggested that nucleoli show at least two basic reaction patterns to inhibitors of RNA synthesis, one typified by actinomycin D and one by ethionine.

Activation of intracisternal A particles in mouse liver by diethylnitrosamine.

Livers of 6- to 7-week-old male C3H/He, CBA, A, and BALB/c mice were examined by electron microscopy for the presence of intracisternal A particles (ICAP) after administration of diethylnitrosamine (DEN) in drinking water. In control mice, ICAP were extremely rare; they were found in the livers of only 2 mice (strains C3H/He and A; none in the other strains). By contrast, the treatment of mice with DEN greatly enhanced the appearance of ICAP in the liver cells of all strains. Within 2 weeks of the treatment, ICAP were found in 8-26% of liver cells examined in all mice and the number of ICAP/cell ranged from 3 to 12. Aside from mild disorganization of the rough endoplasmic reticulum, such as segmentation and vesiculation, liver cells of carcinogen-treated mice showed none of the consistent abnormalities that characterize the appearance of ICAP. The reactivation of ICAP (which are usually suppressed in adult mice) by DEN may become a useful marker for analysis of the sequential alterations of the liver that lead to the development of hepatoma during carcinogenesis.

Relationship of age to prevalence of focal acinar cell dysplasia in the human pancreas.

The prevalence of focal dysplastic lesions of acinar cells in the pancreata of autopsied children and adults was compared. The lesions were recognized in sections stained with hematoxylin and eosin because acinar cells forming islet-sized foci or larger nodules contained one or more of the following cytologic abnormalities: reduced cytoplasmic basophilia, reduced cytoplasm, reduced zymogen, cytoplasmic vacuoles, or nuclear abnormalities. Lesions were found in only 1 patient (age, 7 yr) of 170 patients whose ages ranged from birth to 9 years, whereas 7 of 49 patients 10-19 years old had focal acinar cell dysplasia. The prevalence of such lesions among adults was comparable to that encountered in individuals during the second decade of life and distinctly higher than that found among children during the first decade. Six of the 8 children in whom dysplastic acinar cell foci were found had cancers in other tissues that had been treated by chemotherapy. The data are consistent with the interpretation that dysplastic acinar cell lesions in the pancreas are acquired.

Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.

Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen DL-ethionine.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyltranspeptidase-positive hepatocytes in the liver of carcinogen-treated rats.

The effect of feeding phenobarbital (PHB) with a choline-devoid (CD) diet on the emergence of foci of gamma-glutamyltranspeptidase (GGT)-positive hepatocytes in the liver of carcinogen-treated rats was investigated. Male Sprague-Dawley rats were given a single dose of diethylnitrosamine (50 mg/kg) 18 hr after a partial hepatectomy and 10 days later were placed on a plain choline-supplemented (CS) diet, a plain CD diet, or the CS and CD diets containing 0.06% PHB. Groups of rats were killed after 5 and 7 weeks of feeding each of the four diets, the livers were taken, and the number and size of foci of GGT-positive hepatocytes were determined. In rats fed the CS + PHB diet, the number of foci per sq cm of liver section was greater than that in rats fed the plain CS diet but smaller than that in rats fed the plain CD diet. Addition of PHB to the CD diet resulted in twice as many foci as in the plain CD diet and foci larger than those resulting from the plain CD diet. THe hepatocytes in the foci of rats fed th CD and CD + PHB diets showed, uniformly, not only GGT positively but also a relative absence of fatty change. The results indicate that PHB and a CD diet, when combined, have a synergistic effect in promoting the evolution of liver cells, initiated by a chemical carcinogen, to foci of altered GGT-positive hepatocytes. This promoting regimen may become useful in studies concerned with the initiation and promotion stages of liver carcinogenesis.

Effects of oral versus topical administration of cyclosporine on phorbol ester promotion of murine epidermal carcinogenesis.

In murine epidermal carcinogenesis, topical applications of cyclosporine (CsA), an immunosuppressant, have been reported to suppress 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. In the present study, we compared the effects of p.o. versus topical CsA on TPA promotion of mouse skin tumors and on TPA-induced epidermal hyperplasia. In the first series, groups of male Swiss Webster mice were initiated with 7,12-dimethylbenz(a)anthracene (200 nmol), and 3 days later they were placed on a basal diet or a diet containing 0.015% CsA. Then both groups of mice were promoted twice weekly with TPA (10 nmol) for 22 wk and observed for an additional 13 wk without TPA. No significant difference was observed in the incidence of skin papillomas between the 2 groups. By contrast, the incidences of squamous cell carcinomas in the mice maintained on CsA and basal diet were 67% and 28%, respectively. In the second series, the mice initiated with 200 nmol of 7,12-dimethylbenz(a)anthracene were treated twice weekly with 10 or 5 nmol of TPA for 24 wk. Ten to 15 min prior to each TPA application, one group received topical CsA in acetone (1 mg/mouse), and the other acetone. There was a significant inhibition of TPA promotion in the mice given topical CsA. Topical and p.o. CsA had no significant effect on epidermal hyperplasia induced by 4- to 8-wk treatment of TPA. The mice given topical CsA showed less inflammatory cell infiltrates in the dermis than the mice without CsA. The results indicate that the effect of CsA on TPA promotion of skin tumors depends on its routes of administration, and the p.o. administration enhances the progression of papillomas to squamous cell carcinomas.

Suppression of choline-deficient diet-induced hepatocyte membrane lipid peroxidation in rats by the peroxisome proliferators 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)- acetamide and di(2-ethylhexyl)phthalate.

Many hypolipidemic peroxisome proliferators have been shown to induce liver tumors in rats after long-term feeding. In short-term assays, however, some of them prevent the development of gamma-glutamyl transpeptidase-positive foci, the putative preneoplastic lesions, in the liver of carcinogen-initiated rats and inhibit the promoting effect of a choline-deficient (CD) diet on these lesions. The CD diet-induced lipid peroxidation in the liver has been implicated as one of the underlying mechanisms of the promoting effect. In the present study, the effects of 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamid e (BR931) and di(2-ethylhexyl)phthalate (DEHP) on CD diet-induced liver membrane lipid peroxidation were investigated by determining the extents of conjugated diene formation. No evidence of lipid peroxidation was detected in the microsomal lipids of the liver after administration of BR931 or DEHP at concentrations of 0.16% and 1%, respectively, for 1, 2, and 4 wk. When added to a CD diet, both BR931 and DEHP effectively protected against the diet-induced lipid peroxidation. There was an increase in cellular glutathione levels after 4 wk and an increase in catalase activity after 2 wk in the liver of rats fed BR931 or DEHP. The levels of activity of the glutathione peroxidases and glutathione-s-transferase were significantly reduced. The results suggest that, in the acute stage, hypolipidemic peroxisome proliferator-induced effects of excess production of H2O2 and potential lipid peroxidation are balanced by stimulation of some cellular detoxifying systems. The inhibition of lipid peroxidation by hypolipidemic peroxisome proliferators may account for their inhibitory effects on the CD diet-induced promotion of preneoplastic foci.

Acinar cell carcinoma of the rat pancreas grown in cell culture and in nude mice.

An acinar cell carcinoma of the pancreas, which was induced in a male Wistar rat by repeated injections of azaserine, was propagated in cell culture. Sheets of epithelial-like cells grew within 2 weeks and were subcultured serially. Initially acinar cell carcinoma in the culture medium produced a high level of amylase, but the secretion ceased rapidly as cells began to proliferate. Only negligible amounts of trypsinogen and chymotrypsinogen were detected in cell homogenates at passages 7 and 9. The chromosome distribution ranged from hypodiploid to hypertetraploid. When cultured cells were transplanted s.c. into nude mice, palpable tumors appeared within 4 weeks and could be transplanted serially. Histological examination of the tumor showed poorly differentiated carcinoma without acinar structures. Tumor homogenate contained amylase, trypsinogen, and chymotrypsinogen, and the electron microscopic examination revealed that many tumor cells contained zymogen-like granules. These results indicate that pancreatic acinar cell carcinoma in cell cultures, in which there was no differentiated function, can be activated to synthesize tissue-specific enzymes when transplanted into nude mice by yet undefined factors present in the host animals. The cell line and transplantable tumors of pancreatic acinar cell carcinoma may be useful in the analysis of the biological behavior of this type of tumor and in the study of the control mechanisms of the synthesis of tissue-specific products in cells.

Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.

The effects of feeding a choline-deficient (CD) or a choline-supplemented diet upon the early stages of DL-ethionine carcinogenesis in rat liver were investigated. Low levels of DL-ethionine (0.05 and 0.10%) when fed with a CD diet were found to induce within 4 weeks a massive proliferation of oval cells without significant cell necrosis or presence of inflammatory cell infiltrates. The same levels of ethionine when fed with a choline-supplemented diet caused no significant histological alteration of the liver. In rats fed the CD plus ethionine diets concomitant with the proliferation of oval cells, there was a marked elevation in the content of alpha1-fetoprotein in both liver and plasma. After specific immunofluorescence staining, oval cells stained intensely for albumin and alpha1-fetoprotein. Hepatocytes stained only for albumin, and bile duct cells stained for neither albumin nor alpha1-fetoprotein. These results indicate that a diet deficient in choline markedly alters the response of rat liver to carcinogenetic doses of ethionine. Thus, ethionine hepatocarcinogenesis in rats fed a CD diet may be a useful model for the exploration of the mechanism(s) whereby a dietary factor influences hepatocarcinogenesis.

Stimulation of DNA synthesis and cell proliferation in the liver of rats fed a choline-devoid diet and their suppression by phenobarbital.

Feeding of choline-devoid (CD) diet and dietary administration of phenobarbital (PHB) are efficient promoters of liver carcinogenesis in the rat. Furthermore, inclusion of PHB in a CD diet results in a synergistic effect, inasmuch as the promoting action of their combination is greater than the sum of those exerted by either agent alone. To investigate the mechanism(s) of action of the two promoters, liver DNA synthesis and liver cell proliferation were studied in rats fed a CD diet, a choline-supplemented diet, or the same diets to which 0.06% PHB was added. DNA synthesis was determined by [3H]thymidine incorporation into DNA and autoradiography, and cell proliferation was determined by mitosis counts. Feeding the CD diet caused an increase of both DBA synthesis and cell proliferation over those present in rats fed the choline-supplemented diet. Inclusion of PHB in the CD diet, on the other hand, inhibited DNA synthesis and cell proliferation. These results indicate that stimulation of cell proliferation per se may not be a sufficient condition for an agent to act as a promoter of liver carcinogenesis.

Modulation of epidermal growth factor receptors in rat hepatocytes by two liver tumor-promoting regimens, a choline-deficient and a phenobarbital diet.

The effect of two liver tumor-promoting regimens, a choline-deficient (CD) and a phenobarbital (.06% PB) diet, on the level of epidermal growth factor (EGF) receptor in rat hepatocytes was examined at 3, 10, and 28 days of feeding. Both diets produced a significant decrease in the number of cell surface receptors at 10 and 28 days of treatment. When PB was included in a CD diet, the decrease in the receptor number was evident even after 3 days feeding of the combined diet. Neither diet alone had any effect on the binding at that time. Along with the changes in the receptor number, the binding affinity of EGF to its receptor was also altered by these diets. Furthermore, PB and PB plus CD diets also decreased the EGF binding at the intracellular sites whereas CD diet showed no effects indicating that the decrease in surface binding of EGF by the promoter-treated hepatocytes was not due to rapid internalization of the receptors. The reduced level of hepatocyte surface EGF receptors represents the common property shared by two diverse types of the liver tumor promoters, and may thus be related to the tumor-promoting ability of these agents.

Alterations in hepatocyte insulin receptors in rats fed a choline-deficient diet.

Specific insulin binding and glycogen synthesis were studied in control hepatocytes, hepatocytes from rats fed a choline-deficient (CD) diet for 7 to 14 days, and hepatoma cells induced with a CD diet and DL-ethionine in culture. Both the binding affinity and the number of receptors were affected in hepatocytes by the CD diet. The number of receptor sites was 26,000/cell and the dissociation constant (Kd) for the high affinity binding site was 2.6 nM at 30 degrees C, in contrast to the control values of 205,000 sites/cell and 23.2 nM, respectively. In the hepatoma cells, receptor cell number and Kd were further diminished to 6,400 sites/cell and Kd = 1.1 nM. The basal level of glycogen synthesis in control hepatocytes and in CD hepatocytes was similar; however, the basal rate of glycogen synthesis in hepatoma cells was only 16% of that in the control cells. The glycogen synthesis in hepatoma cells was stimulated by insulin, but at a 3-log higher concentration compared to the control cells. This loss of sensitivity to insulin is consistent with the marked decrease in insulin receptors. CD hepatocytes had a decrease in insulin receptors with a concurrent decrease in Kd (increase in binding affinity), such that, sensitivity to insulin did not differ significantly from that of control hepatocytes. However, the maximal stimulation of glycogen synthesis was only 27% that of the control cells. The changes in receptor number and Kd of hepatocytes from rats fed a CD diet may be due to alterations in cell membrane lipid composition and this alteration may be responsible for the enhanced sensitivity of hepatocytes to chemical carcinogens and for the tumor promoting effect of the diet.

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.

Lipid peroxidation of liver microsome membranes induced by choline-deficient diets and its relationship to the diet-induced promotion of the induction of gamma-glutamyltranspeptidase-positive foci.

The effects of varying the type of dietary fat in the choline-deficient (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of carcinogen-treated rats were investigated, and the results were correlated with the extent of membrane lipid peroxidation induced by the diets. Male Sprague Dawley rats were initiated with a single dose of diethylnitrosamine. Thereafter, groups of rats were fed choline-supplemented or CD diets in which the amount of saturated fat was varied by using hydrogenated vegetable oil (Primex) and corn oil (CO), either alone or in combination. The number and size of GGT-positive foci induced by the CD diet with CO as the sole source of fat were larger than those induced by the diet containing mixtures of Primex and CO. The CD diet with Primex alone was the least effective in inducing GGT-positive foci. Peroxidation of liver microsomal membrane lipids in rats fed regular CD or CD:CO diets was examined by determining the formation of conjugated dienes. The generation of diene conjugate in rats fed a CD:CO diet was evident after 2 days of the diet feeding, and the levels increased at 1 and 2 weeks. No significant diene conjugate was demonstrated in rats fed a regular CD diet for 2 days. However, after 1 and 2 weeks, there was generation of diene conjugate, the levels of which were lower in rats fed the CD diet than those on a CD:CO diet. Addition of an antioxidant, 0.25% butylated hydroxytoluene, to both CD and CD:CO diets abolished the generation of diene conjugate in rat liver microsomal membranes and markedly inhibited the promotion of GGT-positive foci in the liver of diethylnitrosamine-initiated rats. The results suggest that membrane lipid peroxidation in the liver may be related to the promotion of the induction of GGT-positive foci by a CD diet. The enhanced promotion by the inclusion of a higher level of polyunsaturated fat in the diet may be, in part, due to its greater susceptibility to peroxidation.

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.

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.

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.

Lipid composition and 3-hydroxy-3-methylglutaryl-CoA reductase activity of acinar cell carcinoma of rat pancreas.

The lipid composition and 3-hydroxy-3-methylglutaryl-CoA reductase activity of subcutaneous transplantable pancreatic acinar cell tumors on nude mice were compared with those of normal, regenerating, fetal and newborn rat pancreata. The tumors and also the fetal tissues showed decreased concentration in total lipids, increased concentration in sphingomyelin and an increase in cholesterol when compared to normal rat pancreas. The regenerating pancreas showed an intermediate elevation in these lipid parameters. Specifically, only tumor showed an increase in phosphatidylethanolamine/phosphatidylcholine ratio. The tumors and also the fetal tissues showed an increase in hydroxymethylglutaryl-CoA reductase activities, suggesting that the de novo synthesis of cholesterol is a requirement for cell proliferation. The cholesterol metabolism in normal tissues is under metabolic regulation as indicated by decreased hydroxymethylglutaryl-CoA reductase activities and decreased cholesterol concentration in postnatal tissues when compared with the fetal tissues. The fast growing AT3A tumor showed higher hydroxymethylglutaryl-CoA reductase activity when compared to the slow growing AT3B tumor, indicating that the differences in growth rate of the tumors may be related at least in part to differences in their cholesterol metabolism.