Autophagy in Hepatocytes during Distant Tumor Growth.

Structural changes in the liver of CBA mice were studied during the development of experimental hepatocarcinoma-29 inoculated into the hip. A decrease in the volume density of hepatocyte cytoplasm, mitochondria, endoplasmic reticulum, and lipid inclusions and an increase in the volume density of lysosomal structures during tumor growth were observed. All stages of intracellular autophagy were recorded by the method of electron microscopy. These stages included the appearance of autophagosomes, autophagolysosomes, and secondary lysosomes in the hepatocyte cytoplasm. Fragments of cytoplasm, glycogen rosettes, mitochondria, and fragments of endoplasmic reticulum with ribosomes were found in autophagosomes. The obtained data indicate the development of non-selective autophagy in the liver during distant tumor growth in aimed at the maintenance of intracellular homeostasis in hepatocytes and energy and trophic homeostasis of organism.

Changes in cell ultrastructure and inhibition of JAK1/STAT3 signaling pathway in CBRH-7919 cells with astaxanthin.

Astaxanthin (AST), a xanthophylls carotenoid, possesses significant anticancer effects. However, to date, the molecular mechanism of anticancer remains unclear. In the present research, we studied the anticancer mechanism of AST, including the changes in cell ultrastructure, such as the mitochondrion, rough endoplasmic reticulum (RER), Golgi complex, and cytoskeleton, the inhibition of Janus kinase 1(JAK1)/transduction and the activators of the transcription-3 (STAT3) signaling pathway using rat hepatocellular carcinoma CBRH-7919 cells. Cell apoptosis was evaluated and the expressions of JAK1, STAT3, non-metastasis23-1 (nm23-1), and apoptotic gene like B-cell lymphoma/leukemia-2 (bcl-2), B-cell lymphoma-extra large (bcl-xl), proto-oncogene proteins c myc (c-myc) and bcl-2- associated X (bax) were also examined. The results showed that AST could induce cancer cell apoptosis. Under transmission electron microscope, the ultrastructure of treated cells were not clearly distinguishable, the membranes of the mitochondrion, RER, Golgi complex were broken or loosened, and the endoplasmic reticulum (ER) was degranulated. Cytoskeleton depolymerization of the microtubule system led to the collapse of extended vimentin intermediate filament bundles into short agglomerations with disordered distributions. AST inhibited the expression of STAT3, its upstream activator JAK1, and the STAT3 target antiapoptotic genes bcl-2, bcl-xl, and c-myc. Conversely, AST enhanced the expressions of nm23-1 and bax. Overall, our findings demonstrate that AST could induce the apoptosis of CBRH-7919 cells, which are involved in cell ultrastructure and the JAK1/STAT3 signaling pathway.

Chitosan-alginate scaffold culture system for hepatocellular carcinoma increases malignancy and drug resistance.

PURPOSE: Hepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with human HCC cell lines. METHODS: The correlation between the cell function, such as secretion of growth factors and production of ECM in vitro, and the tumor growth and blood vessel recruitment in vivo was investigated. RESULTS: HCC cells grown on 3D CA scaffolds demonstrated morphological characteristics and increased expression of markers of highly malignant cells. Implantation of CA scaffolds cultured with human HCC cells in mice showed accelerated tumor growth. Histology revealed marked differences in morphology and organization of newly formed blood vessels between tumors produced by different pre-cultured conditions. Resistance to doxorubicin was significantly pronounced in CA scaffold-cultured HCC cells compared to 2D or Matrigel cultured HCC cells. CONCLUSIONS: This 3D model of HCC, with its ability to more closely mimic the in vivo tumor behavior, may serve as an invaluable model for study and application of novel anticancer therapeutics against HCC.

Molecular mobility and nucleocytoplasmic flux in hepatoma cells.

Fluorescence microphotolysis (photobleaching) was used to measure, in single polyethylene glycol-induced polykaryons of hepatoma tissue culture cells, nucleocytoplasmic flux and intracellular mobility for a series of dextrans ranging in molecular mass from 3 to 150 kD and for bovine serum albumin. For the dextrans, the cytoplasmic and the nucleoplasmic translational diffusion coefficients amounted to approximately 9 and approximately 15%, respectively, of the value in dilute buffer. The diffusion coefficients depended inversely on molecular radius, suggesting that diffusion was dominated by viscosity effects. By application of the Stokes-Einstein equation, cytoplasmic and nucleoplasmic viscosities were derived to be 6.6 and 8.1 cP, respectively, at 23 degrees C. Between 10 and 37 degrees C nucleoplasmic diffusion coefficients increased by approximately 45-85%, whereas cytoplasmic diffusion coefficients were virtually independent of temperature. In contrast to that of the dextrans, diffusion of bovine serum albumin was more restricted. In the cytoplasm the diffusion coefficient was approximately 1.5% of the value in dilute buffer; in the nucleus albumin was largely immobile. This indicated that albumin mobility is dominated by association with immobile cellular structures. Nucleocytoplasmic flux of dextrans depended inversely on molecular mass with an exclusion limit between 17 and 41 kD. This agrees with previous measurements on primary hepatocytes (Peters, R., 1984, EMBO [Eur. Mol. Biol. Organ.] J. 3:1831-1836), suggesting that in both cell types the nuclear envelope has properties of a molecular sieve with a functional pore radius of approximately 55 A.

Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.

Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels.

Nonhistone protein antigens resolved by electrophoresis in sodium dodecyl sulfate were identified immunochemically after being transferred to nitrocellulose. Use of antiserum to dehistonized chromatin from Novikoff hepatoma revealed numerous protein antigens specific to the chromatin of Novikoff hepatoma in comparison to that of normal rat liver.

Evaluation of centrosome abnormalities and p53 inactivation in chemical induced hepatocellular carcinogenesis.

UNLABELLED: Abnormal centrosome frequently found in human cancer is a major cause of mitotic defects and chromosome instability in cancer cells. Centrosome duplication is controlled in a cell cycle-specific manner, whereas cancer cells with dysregulation of centrosome duplication can survive and reenter the cell cycle through defective cell cycle checkpoint systems. Although numerous studies showed that centrosome amplification can be readily induced by loss or mutational inactivation of p53, however, the role of centrosomally localized p53 in the regulation of centrosome duplication had been enigma. To investigate the role of centrosome and p53 in the in vivo carcinogenesis, we performed immunofluorescence and Western blot analysis, respectively, to detect the alteration of centrosome and p53 status as well as immunohistochemical assay to detect cell proliferation in diethyl nitrosoamine (DENA) induced rat hepatocellular carcinoma (HCC). The frequencies of the centrosome abnormalities in HCC lesions were significantly higher than that of in their preneoplasitc counterparts as well as cell proliferation expression profile. Intriguingly, there was no correlation between centrosome abnormalities and cell proliferation. As for p53, the level of p53 increased in inflammation lesion, but decreased in hepatocirrhosis lesion, even undetectable in HCC lesion. These findings may imply that in inflammatory lesions aberration centrosome occurred irrespective of p53 background. However, the significantly increased percentage of cells with abnormal centrosome in hepatocirrhosis, particularly in HCC lesion concomitant with p53 inactivation and increased cell proliferation rate might synergistically contribute to carcinogenesis. Taken together, centrosome abnormalities were an early event prior to p53 inactivation in the time course of carcinogenesis, suggesting that p53 inactivation may not be the cause of centrosome aberration and centrosome may be a susceptible organelle responding to cellular insults. KEYWORDS: centrosome, p53, hepatocellular carcinoma, cell proliferation.

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 +/- 12.51 vs 170.39 +/- 25.29; 49.83 +/- 11.46 vs 170.39 +/- 25.29; 83.99 +/- 24.63 vs 170.39 +/- 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 +/- 4.2 vs 23.4 +/- 2.1 and 29.4 +/- 3.4 vs 23.4 +/- 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphological changes were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

[Apoptosis induced by the C21 sterols in Baishouwu and its mechanism of action in hepatoma].

This study is to investigate the effect of the C21 sterols on inducing apoptosis of hepatocellular cancer cells and its potential mechanism. The transplanted model of hepatoma substantiality (Heps) was established in mice, and the mice were divided into four groups: negative controls group and C21 sterols groups (10, 20, 40 mg x kg(-1)) , treated with drugs separately once a day for 9 days. Then the mice were sacrificed, the tumor growth inhibition rate (IR) was calculated and tumor tissue samples were taken and examined under electron microscope. The tumor cells were harvested and cell viability or apoptosis was analyzed by acridine orange and ethidium bromide (AO/EB) stain. B-cell lymphoma/leukemia-2 gene (bcl-2) in tumor cells was inspected by immunohistochemistry. After treatment with C21 sterols (10, 20, 40 mg x kg(-1)), inhibitory effect on the transplanted Heps was observed. The IR was 34.79%, 47.08% and 50.23%, respectively. Apoptosis induced by the C21 sterols was observed, low growth density and some apoptotic cells were observed in tumor under the electron microscope. The expression of bcl-2 gene on tumor cells decreased in the C21 sterols groups, but the percentage of positive area is higher in 40 mg x kg(-1) group than that in 20 mg x kg(-1) group, which differed from apoptosis results. Inhibiting the excessive expression of bcl-2 gene to promote apoptosis may be one of anti-tumor mechanisms for the C21 sterols in Baishouwu.

Intermediate-sized filaments in cultured rat liver tumor cells with Mallory body-like cytoplasm abnormalities.

The endoskeletal structure of tumor cells with a characteristic cytoplasmic abnormality initiated in rat liver by the in vivo adminisration of the carcinogen diethylnitrosamine was studied in clonal cell lines established and propagated in vitro. The bulk of the cytoplasmic and plasma membrane protein was removed by extraction with Triton X-100, and subsequently the juxtanuclear detergent-insoluble fraction containing filaments of 100-150 A was released into citrate buffer at pH 2.8. Analysis of this fraction by sodium dodecyl sulfate acrylamide gel electrophoresis revealed the pressence of five major proteins that banded with apparent molecular weights of about 66, 57, 52, 48 , and 43 x 10(3), the last of which comigrated with actin. The proteins thus resembled those from intermediate-sized filaments of both the vimentin (57 x 10(3)) and the prekeratin types obtained from various vertebrate cells. They also appeared to be related to the polypeptides of intermediate-sized filaments from Mallory bodies induced by griseofulvin in the livers of mice and to some of the polypeptides seen in isolates of Mallory bodies from human alcoholics. These results indicated that a major component of the carcinogen-induced lesion consisted of intermediate-sized filaments. The possible significance in cell transformation of this stably maintained aggregate of filaments that binds concanavalin A and displaces the nucleus is discussed. The close resemblance of this lesion to that seen in the cells of cirrhotic livers of alcoholics (Mallory's alcoholic hyalin) raises a question regarding the possible oncogenic status of such cells in humans.

Preparation of plasma membranes from line 10 and line 1 guinea pig hepatomas.

Gentle homogenization followed by differential and density gradient centrifugation was used to purify line 10 and line 1 guinea pig hepatoma plasma membranes in the form of ghosts. Yields of 15--25% allowed enough membranes to be obtained from a single ascites tumor-bearing animal for immunologic and biochemical studies. Although the plasma membrane marker enzyme (Na+ + k+)atpase was present in normal concentrations in both line 10 and line 1 hepatomas, 5'-nucleotidase was reduced over 100-fold in both tumors and phosphodiesterase I was increased 210-fold in the line 10 hepatomas.

Biology of hepatocellular neoplasia in the mouse. III. Electron microscopy of safrole-induced hepatocellular adenomas and hepatocellular carcinomas.

A systematic, ultrastructural analysis wsa performed on safrole-induced hepatocellular adenomas and hepatocellular carcinoma(s) (HPC) in BALB/c mice. Adenomas were heterogeneous in cell composition containing dark-staining basophilic cells, pale-staining acidophilic cells, clear cells, and lipid-laden cells. Darkly staining cells resembled fetal hepatocytes. They had large nuclei with irregular borders and limited diversity of organelles. Rough endoplasmic reticulum was prominent and seen as parallel cisternae in single or double tracts often in association with mitochondria. Pale-staining cells contained abundant smooth endoplasmic reticulum. Other organelles were often displaced to the perinuclear or peripheral region of the cell. The clear cells resembled dark-staining or pale-staining cells but also containing large areas of glycogen deposition. Lipid-laden cells contained numerous, multisized lipid droplets in the cytoplasm. HPC contained cell types similar to those of the adenoma. In addition, they contained many anaplastic cells. These resembled hepatocytes but contained several other alterations. The most striking was an apparent increase in the number of altered mitochondria. The cytoplasm was often fluid with enlarged mitochondria with dense or pale matrices. The cristae were few and had altered configurations. Also, an apparent increase was seen in the number of microbodies. These were often clustered in one region of the cytoplasm. An increase in microbodies was also noted in other cell types of hepatocellular carcinomas. The results of this study demonstrated similarities in the cell types of the adenomas and HPC. This study also demonstrated differences, with the anaplastic cell being common only to the carcinoma. Due to the similarity of cell types, the adenoma should be considered a possible site of HPC development.

Ultrastructural and cytochemical study of the early stages of liver colonization by transplanted neoplastic hepatocytes.

Neoplastic liver cell colonies were induced in the livers of isogeneic F344 rats by intraportal injection of a hepatic cell suspension from diethylnitrosamine-treated donor rats. Examination of the livers 12 days after cell implantation revealed well-demarcated groups of liver cells. The colonies showed alterations of the normal hepatocyte phenotype, which were clearly demonstrated by histologic, cytochemical, and electron microscope techniques. The hepatocytes were markedly deficient in glucose-6-phosphatase and bile canalicular ATPase activities, and they contained numerous mitotic figures. Scanning and transmission electron microscopy allowed characterization of hepatocyte interfaces and the shape of sinusoids and the biliary network. The nodular colonies displayed disorganized, thickened trabeculae separated by dilated sinusoids. In these colonies the hepatocytes proliferated intensely and formed, inside the host parenchyma, revascularized, integrated nodular structures. However, these hepatocytes showed ultrastructural anomalies: large nuclei with prominent nucleoli, many free polysomes, and areas of proliferated smooth endoplasmic reticulum in connection with unfolded cisternae of the rough endoplasmic reticulum. All of these features agreed with the hypothesis previously proposed that the colonies may be precursors of the hepatocarcinomas that ultimately develop in animals given injections of treated liver cells. Direct confirmation, however, still is needed.

Isolation and characterization of chicken liver and hepatoma Mc-29 endoplasmic reticulum and Golgi apparatus membranes and biosynthesis of their glycoconjugates.

Rough and smooth endoplasmic reticulum (ER) membranes and trans-Golgi apparatus (GA) fraction, intermediate GA fraction, and cis-GA fraction from White Leghorn chicken liver and hepatoma Mc-29 were isolated. Their purity was checked by electron microscopy and by determination of the activity of the marker enzymes. Rough and smooth membranes were also identified by calculation of the ratio between the content of RNA and the total phospholipids. It was found that the membrane fractions were pure enough. The tumor exhibited a decreased amount of ER and a small GA when compared when the ER and GA in the liver. The biogenesis of membrane glycoconjugates was followed by in vivo experiments either with [14C]glucosamine or by double labeling with [14C]N-acetylmannosamine and [3H]leucine. The radioisotope studies indicated that the synthesis rate of protein in liver and hepatoma rough ER was nearly the same, but in the tumor cells the glycosylation of the nascent polypeptide chain and the formation of glycolipids were significantly decreased.

Sequential morphologic changes during methapyrilene-induced hepatocellular carcinogenesis in rats.

The pathogenesis of hepatocellular tumors induced in F344 rats by the antihistaminic methapyrilene was investigated by light and electron microscopy in a serial sacrifice study. Eosinophilic foci of altered hepatocytes were found in portal areas after 1 week of treatment, the eosinophilia being caused by proliferation of mitochondria. Eosinophilic neoplastic nodules developed from such lesions after 16 weeks of treatment. Hepatocellular carcinomas developed after 26 weeks of treatment. Mitochondrial proliferation, which had been found as a marker for hepatocytes altered by this compound at 1 week of treatment, was still present in the hepatocellular carcinomas, which therefore met the morphologic criteria of oncocytomas.

Histopathology of neoplastic and nonneoplastic hepatic lesions in mice fed diets containing tetrachlorvinphos.

Tetrachlorvinphos was fed at 8,000 or 16,000 ppm in diets to male and female (C57BL/6N X C3H/HeN)F1 mice for 80 weeks. Surviving mice were killed at 92 weeks, and all mice were completely necropsied. A high incidence of unusual nonneoplastic hepatic lesions in treated mice was present and characterized by pericellular fibrosis, hepatocyte nuclear pleomorphism, and intrasinusoidal foci of macrophages with intracytoplasmic crystalline structures. From 84 to 94% of the treated male mice and from 21 to 23% of the treated females had hepatocellular neoplasms. Only 17% of the control males and 7% of the control females had liver tumors. The induced tumors were frequently multiple in the liver, whereas the tumors in the controls were usually singular. The morphology of 241 liver tumors in 110 treated mice was different from that of tumors in controls. Liver tumors in control mice were generally composed of small basophillic hepatocytes. In treated mice, tumors were hepatocellular carcinomas composed of solid sheets of large basophilic or eosinophilic hepatocytes. Foci of prominent trabecular formation were seen in 51 tumors. Fifteen tumors were composed of small basophilic hepatocytes with oval cells interposed among them. Foci of capillary formation were noted in 3 of these tumors. In addition, 7 more typical hemangiosarcomas forming sinusoids and with thrombosis were observed.

Butyrate-induced cytoarchitectural reorganization of Mallory body-containing rat hepatic tumor cells.

Diethylnitrosamine (CAS: 55-18-5)-transformed 72/22 rat hepatic tumor cells undergo marked cytoarchitectural changes during exposure to sodium butyrate in vitro. Butyrate treatment of this cell line resulted in an increased cell size, volume, and protein content and in structural reorganization within both the intermediate filament and microfilament networks resulting in the generation of a more normal appearing hepatocytic phenotype. Induced changes in the microfilament system involved the accumulation of F-actin at the cellular margins in the form of a peripheral band and in the development of an extensive, predominantly centralized network of thickened cytoplasmic filament bundles. Such butyrate-induced changes in hepatic tumor cellular morphology and microfilament organization were reflected in a 26-51% increase in the amount of cytoskeletal-associated actin in 72/22 cells, as determined by flow cytofluorimetry of permeabilized intact cells or by scanning densitometry of the electrophoretically separated, detergent-resistant cytoskeletal protein fraction, respectively. It is unlikely that this increase in cellular microfilament content was due to a direct effect of butyrate on actin polymerization per se since butyrate (in final concentrations equal to that used in culture) did not alter either actin monomer-polymer transitions or the nucleation reaction in a defined in vitro polymerization assay. The available data suggest that butyrate may regulate the synthesis or modulate the actin-binding capacity of microfilament-associating proteins in cultured cells. Butyrate-induced "normalization" of 72/22 cytoarchitecture was previously shown to be reflected in a reduction or loss in the expression of specific growth traits characteristic of the transformed phenotype. The experimental reversal of defined cytoarchitectural abnormalities and transformed growth characteristics of 72/22 cells by butyrate provided an in vitro model to elucidate both particular cytoskeletal events associated with epithelial cell transformation and the mechanism of action of apparent differentiation-inducing agents, such as sodium butyrate, on responsive tumor cells.