Neoplastic Liver Cells Research Articles

A Morphological Study of Cooling Rate Response in Normal and Neoplastic Human Liver Tissue: Cryosurgical Implications

The process of freezing in normal human livers and in human liver tumors was studied by freezing samples of these tissues with constant cooling rates and then examining the morphology of the frozen tissue, after freeze substitution, with the light microscope. Cooling rates varied from 2°C/min up to approximately 2000°C/min. It was observed that high cooling rates produce extensive intracellular ice in both normal and neoplastic liver. At slow rates of cooling, normal and neoplastic liver cells dehydrated and large extracellular ice crystals formed. Comparison of the frozen normal liver and the frozen malignant tumors shows that for the same rates of freezing, the tumor cells retain more cellular water and therefore show less susceptibility to dehydration at low rates of cooling. At slow cooling rates, the amount of cellular dehydration and consequent vascular and interstitial space engorgement changed with the type of tissue frozen. The greatest amount of dehydration occurred in normal human liver, followed by metastatic colon carcinoma and finally primary hepatocellular carcinoma. These results are important for cryosurgery since they suggest that malignant tissues have a different response to freezing than normal tissues.

Detection of p53 gene mutations in rat hepatocellular carcinoma cell lines by denaturing gradient gel electrophoresis.

Structural alterations of the p53 gene were investigated in chemically induced rat hepatocellular carcinomas (HCCs), hyperplastic hepatic nodules (HPNs), and cell lines derived from rat neoplastic and normal liver cells. The mutations were detected by GC-clamped denaturing gradient gel electrophoresis using DNA that had been amplified from p53 mRNA by the reverse transcriptase-polymerase chain reaction. This method enabled us to find single-base changes within the p53 gene without using radioisotopes. The presence of mutations was subsequently confirmed by DNA sequencing. No mutations were detected in six primary HCCs and 12 HPNs induced by the Solt and Farber regimen (Nature 236: 701-703, 1976), suggesting that p53 gene mutations do not play a major role in rat hepatic carcinogenesis. However, five of seven HCC cell lines and one of two cell lines derived from normal liver cells had the mutated p53 gene and had lost the normal p53 gene. Five cell lines had a G-->T transversion at various codons, whereas one line had a 21-base deletion in exon 5. Therefore, we conclude that p53 gene mutations may occur in vitro during establishment of the cell lines or may be derived from very small populations within the primary tumors.

Expression and distribution of a human colon-carcinoma-associated antigen in normal and diseased liver tissue.

Expression and distribution of 17-1A, a human colon-carcinoma-associated antigen as defined by a monoclonal antibody (17-1A mAb), were evaluated in liver tissues from normal subjects and patients with inflammatory and noninflammatory liver diseases. The antigen recognized by 17-1A mAb is a 41-kD protein that does not belong to the proteins of the cytoskeleton. Using a streptavidin-biotin-peroxidase method on frozen sections of liver tissue, it was located in the cytoplasm of bile duct epithelial cells in normal livers, whereas the hepatocytes were completely unreactive. When diseased liver tissue was examined, a strong 17-1A Ag expression was demonstrable in the epithelium of typical and atypical bile ductules in portal areas. In addition, periportal or periseptal hepatocytes revealed variable staining for 17-1A Ag directly related to acute and chronic inflammatory changes. 17-1A Ag expression in hepatocytes reached the highest frequency in acute hepatitis (5/5) and chronic active hepatitis (17/19). These results indicate that periportal hepatocytes are capable of acquiring antigen expression common to bile ductular cells in inflammatory liver diseases, further supporting the view that these ductules represent transformed hepatocytes. Furthermore, two distinct pictures were found in primary liver malignancies. Neoplastic bile duct epithelium did not maintain 17-1A Ag expression in cholangiocarcinoma, whereas neoplastic liver cells acquired cytoplasmic 17-1A Ag expression in clustered areas in hepatocellular carcinoma and the intensity of staining and antigen distribution were inversely related to the grade of tumor differentiation.

Effects of Modifying Agents on Conformity of Enzyme Phenotype and Proliferative Potential in Focal Preneoplastic and Neoplastic Liver Cell Lesions in Rats

Development of preneoplastic lesions in the rat liver under the influence of various modifiers was investigated with particular attention to changes in simultaneous expression of altered enzyme phenotype within the lesions (conformity) and proliferation potential. Degree of conformity of marker enzymes such as glutathione S‐transferase placental form (GST‐P), glucose‐6‐phosphate dehydrogenase (G6PD), glucose‐6‐phosphatase, adenosine triphosphatase and γ‐glutamyltranspeptidase was compared with levels of S‐bromo‐2‐deoxyuridine labeling. After initiation with diethylnitrosamine, rats were administered the hepatopromoter sodium phenobarbital (PB, 0.05%), the antioxidant ethoxyquin (EQ, 0.5%), or a peroxisome proliferator, clofibrate (CF, 1.0%) or di(2‐ethylhexyl)‐phthalate (0.3%) and killed at week 16 or 32. The PB promoting regimen was clearly associated with increase in the numbers of high conformity class lesions simultaneously expressing three to five enzymes, and elevated proliferation potential. The inhibitor, EQ, in contrast, brought about a time‐dependent decrease in conformity so that only 1 or 2 alterations were most commonly observed at week 32. Lesion populations in the peroxisome proliferator‐ and especially CF‐treated cases were characterized by obvious dissociation between degree of conformity and proliferative status. Such treatment‐dependent differences were not always correlated with the size of the lesion. The results thus suggested that the conformity and proliferation potential of preneoplastic lesions are dependent on modification treatment. Overall, GST‐P was found to be the most reliable marker, although G6PD was less influenced in the peroxisome proliferator cases.

Preneoplastic rat liver cells in vitro: slow progression without promoters, hormones, or growth factors.

In order to determine early changes in liver cells during carcinogenesis and to compare them with normal or neoplastic hepatocytes, an experimental model was established which allowed enrichment of this population at early stages of carcinogenesis and provided sufficient viable material for biochemical and cytogenetic analysis. This paper describes a method that allows in vitro selection and propagation of hepatocytes after in vivo initiation by alkylating agents, without the use of hormones, growth factors, or promoters which might affect their progression. From 6 different rat livers (5 initiated by continuous diethylnitrosamine feeding, 1 by a single exposure to N-methyl-N-nitrosourea) we established slow-growing lines, each of which had its own typical characteristics of growth behavior, morphology, and chromosome number. One of these lines (CL 38) transformed spontaneously after 8 weeks in primary culture, with an abrupt change to typical tumor cell behavior such as focal growth, anchorage independence, cloning ability in soft agar, and tumorigenicity in nude mice and newborn rats. In none of the other lines (now in culture for 11-15 months) has a similar abrupt change yet been observed, but all of them show a steady, albeit slow progression towards the properties of neoplastic liver cells, together with a reduction in chromosome number.

Ultrastructural and Cytochemical Study of the Early Stages of Liver Colonization by Transplanted Neoplastic Hepatocytes<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

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.

The influence of extracellular calcium on the distribution of protein kinase C in non-neoplastic and neoplastic rat liver cells.

Non-neoplastic T51B rat liver epithelial cells cannot proliferate in Ca2+-deficient medium. This proliferative inhibition in Ca2+-deficient medium is accompanied by a large reduction in the amount of cellular EDTA-extractable Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity. By contrast, tumorigenic epithelial cells from several Morris hepatomas proliferate in Ca2+-deficient medium and either maintain or greatly increase their content of EDTA-extractable protein kinase C.

Autonomous cholesterol biosynthesis in murine hepatoma. A receptor defect with normal coated pits

These studies indicate that autonomous cholesterol biosynthesis by hepatocellular carcinoma may result from absent or defective receptors for chylomicron remnants on the surface of the malignant hepatocytes. In vivo, DAB2 hepatoma or liver were perfused with chylomicron remnants labeled with tritiated palmitic acid. Normal liver had chylomicron remnant uptake/gm tissue that was ten times that of hepatoma. In vitro studies using isolated hepatocytes and cultured DAB2 hepatoma cells showed similar results. Uptake of chylomicron remnants labeled with 3H-palmitic acid by normal hepatocytes during a 4-hour period was ten times that of hepatoma cells. Both in vivo and in vitro differences were statistically highly significant (P less than 0.005). Since many surface receptors are related to the coated pits, the cellular membranes of both neoplastic and normal liver cells were examined by electron microscopy. Coated pits were present in both the hepatoma and normal liver cells and occupied 2.61% and 2.65% of the cell surface, respectively. The defective uptake of chylomicron remnants by DAB2 hepatoma appears to be related to the chylomicron remnant receptor and not to the coated pit-internalization mechanism.

Induction of 2′,5′ oligo(A) synthetase in tumor-bearing mice with encephalomyocarditis (EMC) virus of poly(I)poly(C)

Infection of 13 month-old C 3H mice with EMC virus or inoculation with the interferon inducer poly(I)poly(C) results in elevated levels of the enzyme 2′,5′ oligo(A) synthetase only in animals with spontaneous tumors (breast cancer or hepatomas). High enzymatic activities are detected in homogenates from liver, spleen, plasma and neoplastic cells of the animals with breast carcinomas and only in the neoplastic liver cells of the animals with hepatomas.

Evidence That Hepatitis B Virus Has a Role in Liver-Cell Carcinoma in Alcoholic Liver Disease

We compared the presence of serologic markers of hepatitis B virus (HBV) infection with the presence of the viral DNA in the livers of patients with alcoholic liver disease with or without hepatocellular carcinoma. Among 51 patients with various kinds of alcoholic liver disease but without hepatocellular cancer, 19 had one or more serologic markers of HBV, but only three had viral surface antigen in their serum. These three patients, as well as three others who had HBV antibodies but no viral antigen in their serum and two others who had no serologic markers of any kind, had HBV DNA in their liver cells. In at least five of the eight patients with viral DNA in the liver, the DNA was integrated into the genome. Among 20 patients with alcoholic cirrhosis and hepatocellular carcinoma, nine of the 16 tested had serologic markers of HBV infection, but all 20 had HBV DNA integrated into the genome of the neoplastic liver cells. These data suggest that HBV plays a part in the pathogenesis of primary liver-cell cancer in alcoholics.

Application of fine-needle aspiration biopsy for the diagnosis of dysplastic and neoplastic liver cell changes induced by N-nitrosomorpholine in rats.

Male rats were treated with the hepatocarcinogen, N-nitrosomorpholine (NNM, 10 mg ad 100 ml drinking water) for 19 weeks. Repeated fine-needle aspiration biopsies of the liver were performed percutaneously. Cytomorphologic and cytochemical criteria were used for the characterization of dysplastic and carcinoma cells. The alterations seen in the smears were correlated with histopathologic findings in the punctured liver lobes. Cells showing type I dysplasia were recognized in smears obtained from day 7 on. They corresponded to the swollen, glycogen-free cells developing in zone 3 of the Rappaport acinus during the early treatment phases. In later stages type I dysplastic cells were observed in smears. This coincided with the development of neoplastic nodules seen in histopathologic preparations. Carcinoma cells were recognized first after 15 weeks. Marked gamma-glutamyl transpeptidase (gamma-GT) activity could be demonstrated cytochemically in biopsy smears and biochemically in biopsy homogenates during the early phases of NNM-treatment. Simultaneously, a rise in gamma-GT activity was also observed in the serum.

Gamma‐glutamyl Transferase as Oncofetal Marker of Experimental Hepatocarcinogenesis in the Rat

γ‐Glutamyl transferase (γ‐GTase) is abundant in the fetal and neonatal rat liver, but almost completely disappears in the normal adult rat liver. Aflatoxin B1 (AFB1) was used in several hepatocarcinogenic models in the rat. The hepatocarcinogenesis was divided in early, preneoplastic and neoplastic phases during which liver histological changes were studied in correlation with histochemical analysis of γ‐GTase.Five types of hepatocyte populations were encountered: original hepatocytes, small newly formed hepatocytes and transitional cells, early liver cell regenerative areas, late liver cell foci and nodules and evidently malignant liver cells. A very marked γ‐GTase activity always resurged reversibly in every area of proliferating newly formed hepatocytes and transitional cells and in every early regenerative area, but it reappeared irreversibly in every late appearing focus and nodule and in every hepatocellular carcinoma. The whole remaining parenchyma (original hepatocytes) showed no γ‐GTase activity, as in the normal adult rat liver. The resurgence of γ‐GTase in these subsequent cell populations, including the malignant hepatocytes, emphasizes that the development of the latter implicates the reacquiring of some embryonal biochemical characteristics. Furthermore it illustrates the similar immature state of specific and focal preneoplastic and neoplastic liver cell lesions. γ‐GTase histochemically proved to be the most sensitive marker of the hepatocarcinogenic process.

Histological and histochemical studies on the distribution of a few enzymes in the neoplastic liver of Uroloncha malabarica (Linnaeus).

The present work describes histological and histochemical observations made on the neoplastic liver of Indian silver bills, Uroloncha malabarica. The histology of neoplastic tissue as well as liver has been discussed. Further, a few enzymes like alkaline phosphatase, acid phosphatase, 5-nucleotides and non-specific esterase have been localized in the diseased liver. The occurrence of lymphocytoma caused a marked change in the localization of the enzymes. Sometimes total inhibition of the enzyme was encountered. Damaged sinusoid cells and bile canaliculi of the neoplasm as well as liver lobules show no reaction for alkaline phosphatase. However, its counterpart, acid phosphatase, exhibits intense activity in both neoplastic tissue and liver cells. Aggregates of neoplastic tissue give moderate 5-nucleotidase reaction while it gives poor activity in hepatic tissue of the diseased liver. Parenchymatous cells are able to give some activity for the non-specific esterase while it is very dull in the neoplastic tissue.

Increased expression of actin-like contractile protein in preneoplastic and neoplastic lesions in rat liver

Cryostat sections of 16 preneoplastic and 14 neoplastic hepatic lesions induced in rats by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) were examined by indirect immunofluorescence with human serum containing smooth-muscle antibody (SMA). Preneoplastic lesions showed strong cytoplasmic staining of proliferating oval cells and of the cell outlines of hepatocytes, in areas of nodular hyperplasia. In carcinomas, poorly differentiated hepatocytes showed staining of cell outlines, while well differentiated tumour cells forming glandular structures showed only staining of the luminar surfaces. The stromal cells also showed cytoplasmic staining. Morphologically normal areas of 3'-Me-DAB-treated livers showed weak staining of cell outlines, similar to normal liver. Specificity of the staining reactions was established by failure of staining in parallel control sections treated with normal human serum, or SMA serum neutralized by absorptions with homogenates of smooth muscle or extracts of actin. The results suggest that there is an increased expression of actin-like contractile protein in preneoplastic and poorly differentiated neoplastic liver cells.

Surface properties of normal and neoplastic rat liver cells: Lectin-induced cytoagglutination and lectin receptor activity of cell-surface glycopeptides

Rat liver cells were isolated by two techniques: perfusion with citrate followed by mechanical dispersal and enzymatic dissociation by perfusion with collagenase. The viability and ultrastructural integrity of the enzymatically dispersed cells were superior to those dissociated mechanically. Hepatocytes prepared by mechanical dispersal were not agglutinated by high concentrations of concanavalin A (ConA) or wheat germ agglutinin (WGA), whereas hepatocytes isolated by the enzymatic method were agglutinated by both lectins and exhibited a significant increase in their agglutinability after treatment with papain. These data indicate that cell viability and/or structural integrity are required for ConA- or WGA-induced agglutination of rat hepatocytes. A non-dialyzable sialoglycopeptide fraction cleaved from the cell surface by digestion with papain was isolated from enzymatically dispersed hepatocytes and Novikoff ascites hepatoma cells. These sialoglycopeptide fractions were further purified using gel filtration (Sephadex G-50). Comparison of their gel filtration profiles and lectin receptor activities revealed significant qualitative differences between the cell-surface sialoglycopeptides of normal and malignant rat liver cells. The most prominent cell-surface sialoglycopeptide fraction from hepatoma cells was excluded from the gel and possessed the major portion of the lectin receptor activity, whereas the most prominent cell-surface sialoglycopeptide fraction from hepatocytes was partially accessible to the gel and possessed no detectable ConA or WGA receptor activity. These data indicate that papain-labile cell-surface sialoglycopeptides are not involved in the ConA- and WGA-induced agglutination of intact rat hepatocytes and that, in contrast to Novikoff hepatoma cells, ConA and WGA receptor sites are not released by papain digestion of the intact hepatocytes.

35S incorporation into sulfated mucopolysaccharides from embryonic, normal, and neoplastic liver cells

The incorporation of 35S into the sulfated mucopolysaccharides from hepatoma, normal and embryonic liver cells has been studied by means of anion exchange chromatography of the isolated mucopolysaccharide. Comparatively to liver cells, while showing a higher labeling of low sulfated mucopolysaccharides the hepatoma cells incorporate much less 35S into the heparin fraction.

Pyruvate‐Induced changes in hepatoma cell morphology and macromolecular synthesis

AbstractCells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells).Cells were cultured in plalstic T‐flasks (0.5, 1.0, or 2.0 × 106 cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S‐77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10−3, 10−4, or 10−5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate‐enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected.These pyruvate‐induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.

Ultrastructural findings in a well-differentiated hepatoma.

The fine structure of a well-differentiated hepatoma, removed from the liver of a 28-year-old female, was investigated. In the neoplastic liver cells all the organelles were easily recognized. Striking changes were seen in the mitochondria, consisting of polymorphism and increased matrical density. Bizarre-shaped giant mitochondria were formed, containing paracrystalline inclusionsin their internal compartment. The genesis of mitochondrial abnormalities is discussed.

Ethionine induced sRNA neomethylase activity

Recent studies in this laboratory have been directed towards the methylation of sRNA using adult, embryonic, and neoplastic mouse liver cell preparations (Hancock, 1966; Hancock, 1967a; Hancock, 1967b; Hancock, et al. , 1967) . After characterizing the sRNA ethylase activity of mouse liver (manuscript submitted) investigations were initiated to detect any changes in sRNA ethylase activity of ethionine treated mice. Although the sRNA ethylase activities thus far are not remarkable, it was discovered during experiments analyzing rates of methylation that sRNA methylase activity had definitely increased in livers from ethionine treated mice. This paper will present evidence that new cytidylate, adenylate, and uridylate sRNA methylase activities arise in the liver cells of ethionine treated mice. Only data concerned with mice fed ethionine for six months will be presented here and a full report including three month data will appear elsewhere after the analysis of mice fed for nine months with an ethionine diet and completion of sRNA ethylase experiments.

Electrophoretic Mobilities of Normal and Regenerating Liver Cells

BY measuring the electrophoretic mobility of cells, Ambrose, James and Lowick1 have demonstrated that the average charge density of neoplastic kidney and liver cells is almost double that of the homologous normal cells.