Phenotypic reversion of rat neoplastic liver nodules is under genetic control

Low DNA synthesis and high redifferentiation (remodeling) characterize neoplastic nodules induced by chemical carcinogens in hybrid BFF1 rats, generated by crossing the susceptible F344 and resistant BN strains. We performed whole-genome scanning of BFF2 rats to identify loci controlling remodeling of nodules induced, 32 weeks after initiation with diethylnitrosamine, by the RH protocol. Remodeling nodules were identified as areas lacking uniformity of GST-P immunostaining and with irregular margins. Two loci in suggestive linkage with the percentage of remodeling nodules were identified on chromosomes 7 and 1 (LOD scores 3.85 and 2.9 at D7Rat25 and D1Mgh14). Significant dosage-negative effect of the B allele on remodeling and additive interaction between these loci were found. Significant epistatic interactions, showing a recessive, remodeling-enhancing effect of B alleles, occurred between D1Mit3 and D11Rat11 (corrected p = 0.0013) and between D6Rat14 and D8Rat46 (corrected p = 0.028). These data show that remodeling of neoplastic nodules during rat hepatocarcinogenesis is under genetic control. Loci affecting remodeling map to chromosomal regions syntenic to chromosomal segments of human HCC showing structural abnormalities.     

Persistence and growth of rat liver neoplastic nodules following cessation of carcinogen exposure.

Altered (hyperplastic) foci and neoplastic (hyperplastic) nodules identified by their resistance to iron accumulation were induced in the livers of F344 rats by limited feeding of N-2-fluorenylacetamide for three, four, or five cycles of 4-week intervals separated by 1 week of a basal diet. Foci were present by the end of three feeding cycles and increased in number with further carcinogen exposure. No nodules were present at the end of three or four cycles, but they appeared at later intervals after removal of the carcinogen. Nodules were present at the end of five cycles of feeding and increased in number later. Thus nodules were found to be persistent and to have the progressive growth ability in situ that is characteristic of neoplasms.     

Asialoglycoprotein receptors in rat liver nodules

The asialoglycoprotein (ASGP) receptor, binding and internalizingglycoproteins exposing terminal galactose or N-acetylgalactosamineresidues, was compared in normal liver, regenerating liver andliver nodules. The total cellular content of ASGP binding siteswas reduced to 50 and 40% in regenerating liver and liver nodulesrespectively, compared to the level in normal liver. The ASGPreceptor was heterogeneously distributed in subcellular fractions,with the highest enrichment (16-fold) found in a low-densitymembrane fraction (LDMF), enriched in endosomes and Golgi complexmembranes. The subcellular distribution pattern was similarin the three different liver tissues, except that the relativeenrichment in LDMF was less pronounced in regenerating liver(13-fold) and even less in liver nodules (5-fold). Scatchardanalysis of the binding data indicated that the receptor populationsin all liver tissues were homogeneous with dissociation constantsin the range of 0.12-0.47 nM. The difference in ASGP receptorbinding activity was not found to be the result of an increasedoccupancy with endogenous ligand. In vivo endocytosis of [¹²⁵I]asialo-orosomucoid([¹²⁵I]ASOR) showed a reduction in the amount of internalizedligand in liver nodules, well correlated with the reduced numberof binding sites compared to normal liver. However, a slowerthan normal intracelhilar metabolism of internalized ligandin the nodules was indicated. Bifunctional cross-linking experimentsshowed [¹²⁵I)ASOR-receptor complexes of M_r 250 000, 110 000and 85 000 in normal and regenerating liver, whereas in livernodules only the M_r 85 000 was seen. It is concluded that ASGPbinding activity is reduced in regenerating liver and in livernodules. This reduction was manifested predominantly in membranesderived from the Golgi complex, in endocytic vesicles and atthe cell surface. The slower than normal rate of decay of theinternalized ligand in liver nodules is suggested to be theresult of alterations in ligand dissociation and receptor recyclingprocesses. Furthermore, the abscence of high molecular cross-linkable[¹²⁵I]ASOR-receptor complexes in liver nodules may reflect analteration in receptor oligomerization.     

Phenobarbital specifically reduces gap junction protein mRNA level in rat liver

The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm-associated marker genes glutathione S-transferase (placental form) and gamma-glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital-mediated liver tumor promotion.     

Enhanced survival and absence of progressive growth of transplanted rat liver altered eosinophilic foci and neoplastic nodules in phenobarbital-treated rats

Hepatocellular altered eosinophilic cell foci and neoplastic nodules induced in inbred F344 male albino rats by the feeding of N-2-fluorenylacetamide were transplanted into the inguinal mammary fat pads of syngeneic animals to assess their ability to produce neoplasms. Treatment of one-half of the recipients with the neoplasm promoter phenobarbital sodium enhanced survival of both types of transplants. However, at the end of 6 or 12 months of maintenance of recipients of altered foci and 6 months of maintenance of recipients of early and late nodules, no neoplasms occurred, in spite of persistence of abnormal cells at the transplant sites. Therefore, the conclusion was that these carcinogen-induced liver lesions possess limited growth capacity in this ectopic site and that transition to carcinoma under these conditions did not occur in high frequency.     

Short-term induction of neoplastic nodules in the rat liver. II. Study of their development and the effects of withdrawal of 2-acetylaminofluorene

Neoplastic liver nodules were induced by a single administrationof N-nitrosodiethylamine (NDEA) and selectively stimulated togrowth by 2-acetylamino-fluorene (2-AAF) and partial hepatectomyto provide morphological data on very early and late stagesof pre-neoplastic development. Presumptive preneoplastic livercells were recognizable by light and electron microscopy by2 days after partial hepatectomy, and they developed within2 weeks into large, solid nodules consisting of plates of 2or 3 cell layers thick, that compressed the surrounding non-nodulartissue. The cells showed nuclei with an enlarged nucleolus,tortuously dispersed rough endoplasmic reticulum (RER) and anelectron lucent cytoplasm. These features remained present throughoutthe first 2 weeks of growth. During this period the initiallysmall, distinct smooth endoplasmic reticulum areas proliferatedgradually. They were associated with an increasing incidenceof cytoplasmic membranes whorl formation and incidently withannulate lamellae. After withdrawal of 2-AAF the majority ofthe nodules regressed resulting in slowly disappearing fociof glycogen rich cells. A relatively small number of solid nodulespersisted. They were characterized by large cells with a homogeneouseosinophilic cytoplasm, enlarged nucleoli and dispersed RER.Because these characteristics were absent in nodular cells thathad reverted to normal liver cells, it is not appropriate toconsider these characteristics to represent neoplastic transformationper se.     

Flow cytometric analysis of neoplastic nodules and hepatocellular carcinomas induced by ciprofibrate in the rat.

Alterations in DNA ploidy accompany hepatocellular carcinoma (HCC). However, changes in DNA content are also seen in regenerating liver and with increasing age. Thus, to investigate the role of DNA ploidy changes in development of HCC, flow cytometric DNA content determinations were done in a rat model system of peroxisome proliferator-induced HCC. Paraffin blocks of liver isolated from 18 Fisher 344 male rats fed ciprofibrate for 20 weeks (4), 40 weeks (4) or 20 months (10) were examined. Livers from age-matched control rats were also examined. From the 20 month ciprofibrate group, nine neoplastic nodules (NNs), 27 HCCs and four non-tumorous surrounding tissue controls (NTCs) were examined. Significant DNA tetraploid populations were seen in both the NNs and NTCs. A significant increase in the percentage of DNA diploid cells was observed in the NN samples. No significant difference in the percentage S-phase cells was seen. Emergence of cell populations with new DNA ploidy classes (8c or DNA aneuploid) as compared with NTCs was only seen in HCCs (7 of 27), and five of these seven were DNA aneuploid, as distinct from DNA tetraploid, populations. A total of 16 of 24 HCC samples that were adequate for cell cycle analysis had average percent S-phase greater than the mean of the NTCs plus three standard deviations. Although a direct role cannot be inferred, these results support the hypothesis that increases in the fraction of diploid cells is an important early event in the development of rat HCC and that further alterations in DNA ploidy and increased proliferative fraction accompany the development of HCC.     

Heme enzyme patterns in rat liver nodules and tumors

Chemically induced rat hepatocyte nodules and carcinomas have a reduced capacity to oxidize drugs. The reduction in monoxygenase activity results largely from the partial loss of cytochrome P-450, a heme-containing terminal electron acceptor. To determine whether the cytochrome P-450 deficit was indicative of an altered heme metabolism, we quantitated four heme-containing proteins in normal rat liver and in rat liver nodules and cancers induced by 2-acetylaminofluorene or diethyl-nitrosamine: cytochrome P-450; cytochrome bs; catalase (EC 1.11.1.6); and tryptophan 2,3-dioxygenase (EC 1.13.11.11). The amounts of these components in nodules were 45%, 88%, 50%, and 59% of normal liver, respectively; in 2-acetylaminofluorene-induced cancers, 65%, 74%, 64%, and 65%, respectively; and in diethylnitrosamine-induced cancers, 40%, 69%, 56%, and 52%. delta-Aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in nodules were 95% and 138% of normal liver, respectively, whereas in 2-acetylaminofluorene-induced cancers, they were 47% and 233%, and in diethylnitrosamine-induced cancers, they were 50% and 175%. These data indicate that four nonmitochondrial liver hemoproteins were diminished to about the same extent in hepatic nodules and cancers. Nodules and cancers also demonstrated an increased capacity for heme degradation, while cancers also demonstrated a decreased capacity for heme synthesis. Thus, the resistance of nodules and tumors to P-450-activated cytotoxic agents may ultimately result from a disturbance in heme metabolism.     

Inositol phosphates and phosphoinositides in rat liver nodules.

Total amounts and turnover rates of phosphoinositides and inositol phosphates in normal rat liver and hepatocyte nodules were investigated. Male Wistar rats were injected i.p. with [3H]inositol 18-20 h before killing. The amount of phosphatidylinositol in a homogenate preparation was roughly doubled in the nodules, though levels of polyphosphoinositides were approximately the same. Basal levels of inositol phosphates were the same in nodules and in normal liver. Turnover rates of inositol tris- and tetrakisphosphates were studied after stimulation of intact cells with vasopressin for different periods of time (0-5 min). The initial rate of formation of inositol trisphosphate after agonist exposure was fast in both nodular and normal cells. Nodular cells reached peak amount of inositol trisphosphate at 2.5-fold basal levels after 20 s, while normal cells peaked after 40 s at 4.5 times the basal amount. The level of inositol tetrakisphosphate was enhanced very quickly in normal cells, but in the nodular cells there was no increase of this inositol phosphate after vasopressin stimulation. To investigate the mechanism of this difference, the activities of inositol 1,4,5-trisphosphate kinase and of inositol 1,4,5-trisphosphate phosphatase were studied. Both activities were rapid and equal in nodules and normal liver. The amount of cell surface receptors for vasopressin was shown to be one-third in the nodules, as compared to normal cells. This quantitative decrease in receptor number was reflected in lower formation of inositol trisphosphate when stimulated with vasopressin, but could not explain the loss of inositol tetrakisphosphate response in nodules. The significance of the reported alterations in second messenger traffic for the growth regulation of nodular cells and for their progression to carcinoma is not yet known, but could add to the nodules being less dependent on growth regulating signals.     

The PPARgamma agonist pioglitazone inhibits early neoplastic occurrence in the rat liver

Hepatocellular carcinoma (HCC) is increasing worldwide and is the fifth main cause of cancer-related death. HCC develops on a preneoplastic organ, the cirrhotic liver. Therefore, chemoprevention could play a role in the therapy of HCC. We evaluated the preventive effects of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the induction of early carcinogenic events. We monitored pre-neoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in rats, using diethylnitrosamine and acetylaminofluorene. Pioglitazone treatment was initiated the day after the first diethylnitrosamine injection. By quantitative morphometry and Western blot, we showed that pioglitazone significantly decreases the size of pre-neoplastic foci. Analysis of proliferation and apoptosis, assessed by immunohistochemistry, demonstrated decreased proliferation but no effect on cell death in rats treated with pioglitazone. These events were associated with an increased expression of the cyclin-dependent kinase inhibitor p27(kip1), compared to the non treated group. In conclusion, pioglitazone inhibits early carcinogenic transformation in a two-step rat model. As pioglitazone has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC in humans in a clinical setting.     

Enhanced O6-methylguanine-DNA alkyltransferase activity in rat liver nodules

The progression of chemical carcinogen induced hepatic lesions in the rat from altered enzyme foci to hepatocyte nodules and ultimately to hepatocellular carcinoma appears to be related to the evolution of new cell populations within these hepatic lesions. Initiator-promoter-initiator experiments in rat liver models using alkylating agents as the second genotoxic compound suggest accelerated progression toward malignancy could be the result of mutations caused by O6-alkylguanine formation in the DNA of preneoplastic liver cells. Since mutation frequency is believed to be related not only to the extent of O6-alkylguanine formation in DNA, but also to the rate of O6-alkylguanine repair, we measured the activity of the enzyme which repairs O6-alkylguanine, O6-alkylguanine-DNA alkyltransferase, in rat liver nodules. The activity of O6-methylguanine-DNA alkyltransferase in extracts of rat liver nodules 15 and 25 weeks post-initiation was approximately 1.4- and 1.8-fold greater, respectively, than comparable extracts from untreated-control and promoter only-treated rat liver tissues. Thus, the enhanced progression toward malignancy caused by treatment of rats bearing carcinogen-induced preneoplastic hepatic lesions with alkylating agents cannot be explained by a generalized deficiency of O6-alkylguanine-DNA alkyltransferase in hepatic preneoplastic lesions as a whole. These studies show that the distinctive xenobiotic resistant phenotype of rat hepatocyte nodules includes, in addition to the previously documented alterations in xenobiotic activation and detoxification enzyme activities, enhanced activity of a specific DNA repair system.     

Decreased vacuolar acidification capacity in drug-resistant rat liver preneoplastic nodules

Rat liver nodules produced by intermittent 2-acetylaminofluorene feeding exhibit alterations in cell surface receptors reminiscent of impairment of vacuolar acidification. In this report, vacuolar acidification activity, measured as the ATP-dependent quenching of acridine orange, was characterized in liver and nodular membrane fractions using various ion-transport inhibitors and with respect to nucleotide specificity and divalent cation dependence. Based on these criteria and on the comparison of vacuolar acidification activity with mitochondrial, lysosomal, and plasma membrane marker enzymes in different subcellular fractions, it was concluded that the assay measures the proton pump associated with exocytic and/or endocytic vacuolar compartments. When the vacuolar acidification activity was compared in liver and nodular subcellular membrane fractions, it was found that the vacuolar acidification was most strongly reduced in nodular low-density membrane fractions enriched in Golgi-derived membranes and endocytic vesicles. The data indicate that vacuolar, i.e., exocytic and/or endocytic, prelysosomal intracellular compartments in rat liver nodules are markedly deficient in acidification capacity, possibly providing an explanation to various metabolic aberrations, such as diminished iron accumulation and reduced protein degradation, observed in rat liver nodular cells.     

Analysis of loss of heterozygosity in neoplastic nodules induced by diethylnitrosamine in the resistant BFF1 rat strain.

Loss of heterozygosity (LOH) at specific chromosomal regions is a frequent event in poorly differentiated human hepatocellular carcinomas (HCCs), but rare in mouse HCCs. This behavior could depend on interspecies differences in mechanisms of hepatocarcinogenesis or in developmental stage of lesions. To verify if LOH is involved in rat hepatocarcinogenesis, we studied LOH frequency in slowly growing neoplastic nodules induced by Solt-Farber model in diethylnitrosamine-initiated BFF1 rats. We analyzed, with microsatellites, markers at 67 rat loci dispersed over all chromosomes, corresponding to regions homologous to those lost in human HCCs or containing hepatocellular susceptibility (Hcs) or resistance (Hcr) loci in rat and mouse. In agreement with previous findings with mouse HCCs, but at variance with human HCCs, no detectable LOH was found at any locus in rats, suggesting rare LOH involvement in neoplastic nodules, with low tendency to progress to full malignancy, of BFF1 rats.     

Growth regulation and tumour formation of normal and neoplastic rat cells

The oncogenicity of cells in syngeneic hosts was compared to the regulation of cell growth in vitro. Normal embryonic fibroblasts (LWF cells) were not tumorigenic. Spontaneously “transformed” fibroblastic cells (LW13 cells) and Rous-transformed epithelioid cells (RsK4 cells) grew into sarcomas on subcutaneous or intravenous inoculation. TD₅₀ varied according to the route of administration and dispersion of the cells. Rous-transformed cells gave rise to tumours with a short latent period while the spontaneously transformed cell line showed a biphasic latent period. The histopathology of the two kinds of tumours was similar, with a higher tendency to pleomorphism in the Rous tumours. Normal fibroblasts had low plating efficiency on plastic or in soft gel suspension. LW13 cells plated with high efficiency on plastic but poorly in suspension, and RsK4 cells plated with high efficiency in both situations. Normal cells were highly sensitive to topoinhibition, LW13 cells were moderately sensitive, while LW13 cells explanted from tumours and RsK4 cells were the least sensitive. However, values of topoinhibition and its compensation by serum were themselves found to vary with cell density. RsK4 cells initiated DNA synthesis in low serum medium but normal cells and LW13 cells did not. Neither LW13 nor RsK4 cell proliferation was inhibited by contact with confluent, static 3T3 cells or normal LWF cells. With the cell types examined here an association exists between contact inhibition of cell movement and topoinhibition of growth. The degree to which altered cells respond to the regulatory contact signals of normal cells appears to be the best in vitro indicator of oncogenicity.     

Implications of reactive oxygen species on cancer formation and its treatment

Elevated levels of reactive oxygen species (ROS) are a hallmark of cancer. Although increased ROS concentrations play important roles in cancer formation and progression, levels above a cytotoxic threshold cause cancer cell death. Cancer cells adapt to high concentrations of ROS via antioxidant production and reprogrammed cellular metabolism (eg, the Warburg effect). Because some widely used anticancer therapies such as radiation therapy and chemotherapy rely on ROS accumulation as a mechanism to induce cancer cell death, a cancer cell's ability to control ROS levels is a driver of treatment resistance and a critical consideration for successful cancer treatment. The necessity for cancer cells to adapt to elevated levels of ROS to survive may represent an Achilles heel for some malignancies, as therapies designed to interfere with this adaptation would be expected to kill cancer cells. In this review, we provide an overview of the implications of ROS on cancer formation and anticancer treatment strategies, with a focus on treatment-resistant disease.     

The galactosyltransferase activity of hepatic nodules during rat liver carcinogenesis

Galactosyltransferase has been isolated from putative preneoplastic hepatocyte nodules generated in the resistant hepatocyte model by the procedure of Solt et al. (Am. J. Pathol., 88 (1977) 595-609). The following observations have resulted from these studies: (a) the specific activity of galactosyltransferase isolated from hepatocyte nodules by affinity chromatography was reduced to about 1/3 that of the enzyme in control and in liver tissue surrounding the nodules; (b) the galactosyltransferase activity from normal rat serum eluted from the alpha-lactalbumin affinity column as a single peak (spec. act. = 1.57 nmol/min per mg) while that from the serum of nodule-bearing rats eluted in two distinct peaks (spec. act. = 2.49 and 0.49 nmol/min per mg protein); (c) the elution profile of the enzyme from hepatocyte nodules was broad compared to that from normal liver, surrounding liver or serum; (d) the Km for N-acetyl-D-glucosamine (GlcNAc) was lower in all four independent batches of nodules compared to the Km for GlcNAc from control and surrounding liver; (e) the Km for uridine diphosphogalactose (UDP-Gal) was higher for the enzyme from nodules compared to that from control tissue. These data suggest that the hepatocyte nodule produces several glycoforms of galactosyltransferase the kinetic properties of which differ from those of the enzyme from control liver.