Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 ± 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.     

GDP-fucose:GM1 alpha 1----2fucosyltransferase is activated in parenchymal cells of rat liver during early stages of N-2-acetylaminofluorene induced hepatocarcinogenesis

Gangliosides from liver parenchymal and non-parenchymal cells were isolated from Fischer 344 rats that had been fed normal diet or a diet supplemented with 0.03% N-2-acetylaminofluorene (AAF) for 4 weeks. Gangliosides from liver cell fractions were characterized by an induction of both II3NeuAcIV3 alpha GalIV2FucGg4 and GM3 synthesis in the parenchymal cells of AAF-fed animals which were missing in parenchymal cells from animals fed normal diet. In addition, new bands corresponding to GM1 and GD1a were observed in cell fractions of AAF-fed animals. The activity of the GM1-specific alpha 1----2fucosyltransferase induced after AAF feeding was found to be enriched 5- to 6-fold in the parenchymal cell fraction of AAF-fed animals and correlated with the parenchymal cell marker enzyme glucose-6-phosphatase in these cell fractions. Feeding animals the hepatotoxin acetaminophen at 1.87% in the diet for 10 weeks resulted in no increase in the levels of the alpha 1----2fucosyltransferase. Antibodies specific for II3NeuAcIV3 alpha GalIV2FucGg4 were produced and utilized in tissue localization studies. These results indicated little or no staining of normal liver tissue or that after acetaminophen feeding was observed. In contrast, focal areas of staining of liver tissue from animals after 3 weeks of 0.03% AAF feeding were readily apparent. These results indicate that induction of alpha 1----2fucosyltransferase and fucoganglioside synthesis is most probably a property of liver parenchymal cells and is associated with events occurring during early stages of AAF-induced carcinogenesis.     

Changes in alanine transport in plasma membrane vesicles from rat liver during the early stages of diethylnitrosamine-induced hepatocarcinogenesis.

The transport of L-alanine, a natural substrate of system A, across liver plasma membrane vesicle preparations was modified during the early stages of rat DENA hepatocarcinogenesis. Kinetic studies indicated an increase of the Vmax, with normal Km values, at 30 h in rats undergoing a partial hepatectomy. Normal Vmax and drastically reduced Km values were present using membrane preparations from liver tissue showing enzyme-altered hyperplastic foci and/or preneoplastic nodules. The results suggest that alanine transport is differently affected by initiating and promoting stimuli during rat DENA hepatocarcinogenesis. The changes of the Vmax could be related to the promoting effect of partial hepatectomy on cell proliferation whereas the changes of the affinity constant (Km) could be the result of intrinsic modifications of the transporter in initiated cells.     

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.     

Evidence for ras gene mutation in 2-amino-3-methylimidazo[4,5-f]quinoline–induced colonic aberrant crypts in the rat

Aberrant crypt foci (ACF) are putative peneoplastic lesions that develop after treatment of animals with colon carcinogens, including cooked-meat heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 rats given IQ by gavage on alternating days for 2 wk (130 mg/kg body weight) and killed 12 wk after the final carcinogen dose had an average of 4.4 ACF/colon and an average of 3.2 crypts/focus. The DNA from these ACF was amplified by the polymerase chain reaction and analyzed by 3′-primer mismatch and direct sequencing methods for mutations in the Ki-ras proto-oncogene. Of the 37 IQ-induced ACF screened, three contained a GGT→GAT mutation in codon 12 and one contained a GGC→GCC mutation in codon 13. The approximately 11% frequency of mutation in IQ-induced ACF is within the range of previous ACF studies of azoxymethane, which reported a 7–37% incidence of Ki-ras mutaion. These findings suggest that for both compounds, ras mutations occur during early stages of colorectal tumorigenesis. However, while ras mutations can be detected with increasing frequency in azoxymethane-induced adenomas and carcinomas, they are reportedly absent in IQ-induced colon tumors. Thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis. © 1995 Wiley-Liss Inc.     

Down-regulation of the microRNAs miR-34a, miR-127, and miR-200b in rat liver during hepatocarcinogenesis induced by a methyl-deficient diet

Altered expression of microRNAs (miRNAs) has been reported in diverse human cancers; however, the down-regulation or up-regulation of any particular miRNAs in cancer is not sufficient to address the role of these changes in carcinogenesis. In this study, using the rat model of liver carcinogenesis induced by a methyl-deficient diet, which is relevant to the hepatocarcinogenesis in humans associated with viral hepatitis C and B infections, alcohol exposure and metabolic liver diseases, we showed that the development of hepatocellular carcinoma (HCC) is characterized by prominent early changes in expression of miRNA genes, specifically by inhibition of expression of microRNAs miR-34a, miR-127, miR-200b, and miR-16a involved in the regulation of apoptosis, cell proliferation, cell-to-cell connection, and epithelial-mesenchymal transition. The mechanistic link between these alterations in miRNAs expression and the development of HCC was confirmed by the corresponding changes in the levels of E2F3, NOTCH1, BCL6, ZFHX1B, and BCL2 proteins targeted by these miRNAs. The significance of miRNAs expression dysregulation in respect to hepatocarcinogenesis was confirmed by the persistence of these miRNAs alterations in the livers of methyl-deficient rats re-fed a methyl-adequate diet. Altogether, the early occurrence of alterations in miRNAs expression and their persistence during the entire process of hepatocarcinogenesis indicate that the dysregulation of microRNAs expression may be an important contributing factor in the development of HCC.     

Molecular mechanisms of TPA-mediated inhibition of gap-junctional intercellular communication: evidence for action on the assembly or function but not the expression of connexin 43 in rat liver epithelial cells.

We found that a rat liver epithelial cell line (IAR 20) expresses connexin 43, the major cardiac gap-junction protein, but not connexin 26 or connexin 32, major liver gap-junction proteins. The effects of TPA on connexin 43 expression in IAR 20 were investigated using northern blot analysis, western blot analysis, and an immunofluorescence technique. Gap-junctional intercellular communication (GJIC) in this cell line decreased within 60 min of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment and recovered after 24 h. The number of immunofluorescence spots of connexin 43 on IAR 20 was closely related to the change in GJIC induced by TPA. However, TPA did not change the level of mRNA measured by northern blot analysis. Moreover, connexin 43 protein expression analyzed by western blotting suggests that connexin 43 proteins were still present in TPA-treated cells at a similar level. These results suggest that GJIC of these rat liver epithelial cells was mediated by connexin 43 protein and that TPA inhibited GJIC by inhibiting posttranslational processing of connexin 43 proteins, e.g., localization or assembly.     

Direct regulation of transforming growth factor β‐induced epithelial–mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells

Transforming growth factor β (TGFβ) causes the acquisition of epithelial–mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ‐induced phosphorylation of its C‐terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C‐terminus (PTEN4A), but not PTEN wild, inhibits TGFβ‐induced EMT. Nevertheless, whether or not the blockade of TGFβ‐induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C‐terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ‐induced EMT in lung cancer cells. The unphosphorylated PTEN C‐terminus might not directly retain the phosphatase activities and repress TGFβ‐induced EMT; the modification that keeps the PTEN C‐terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ‐induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C‐terminus might be a therapeutic target to negatively regulate TGFβ‐induced EMT in lung cancer cells.