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.

Membrane recycling after endocytosis of cationized ferritin in cultured rat hepatocytes.

Cultured hepatocytes from adult rat livers were incubated for various durations (2 to 120 min) with cationized ferritin (CF) to trace the fate of surface membrane. CF was taken up by endocytosis in clathrin-coated vesicles and was found within 15-60 min in endosomes and mainly in multivesicular bodies (MVB). Lysosomes exhibited but scanty labeling, and the marker was not found in Golgi cisternae even at the longer incubation time. Two main observations result from our ultrastructural study: smooth vesicles seem to pinch off from the MVB to move toward the cell surface; moreover, smooth or coated vesicles fuse with secretion vesicles and recycle back to the plasma membrane. The present data thus demonstrate in cultured hepatocytes, the existence of intracellular pathways of plasma membrane that short-cut the Golgi cisternae and the lysosomal compartment.

Invasiveness of hyperplastic nodule cells from diethylnitrosamine-treated rat liver.

Administration of diethylnitrosamine p.o. to female Sprague-Dawley rats induces hepatocellular lesions antecedent to hepatocarcinoma (altered foci and hyperplastic nodules). We have tested hepatocytes from hyperplastic nodules for their invasiveness in vitro, which is a marker for malignancy. The hyperplastic nodule cells are compared with control liver cells and hepatocarcinoma cells. Control liver tissue and the hepatocarcinoma are collected as fragments taken directly from the rat liver. Nodules, on the other hand, isolated by collagenase perfusion of the liver, are collected on a filter. The fragments of normal liver, the nodules, and the hepatocarcinomas were brought in contact with precultured 9-day-old embryonic chick heart fragments for attachment to each other to form confronting cultures. After attachment, the confronting cultures are incubated at 37 degrees on a gyratory shaker for 24 hr to 14 days. Hepatocytes from the nodules show progressive invasion into the precultured heart fragments in the same way as the hepatocarcinoma cells after 3 to 14 days in vitro. The control hepatocytes from the liver fragments showed no invasion. We conclude from these observations that the cells of the nodules must be considered as malignant altered hepatocytes, for they show progressive invasiveness in vitro.

Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes.

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.

[In hospital and at home parenteral nutrition with a single unit (author's transl)]. / Nutrition parentérale en récipient unique chez des patients hospitalisés et à domicile.

Parenteral administration of a nutritive mixture in a disposable bag prepared in a specialized center reduces the incidence of complications and allows for ambulatory treatment at home. The authors use a nutritive mixture of glucose solutions, amino-acids and lipids in a single bag. Turbidity studies, morphometric evaluation, the macroscopic aspect of the emulsions as well as bacteriological studies have demonstrated the stability of the solution for 5 days when kept at 4 degrees C. Forty-nine cancer patients benefited by this therapeutic modality. Four patients developed an infection in conjunction with the advent of temporary personnel in specialized unit. One patient developed cholestatic jaundice. All 5 recovered. No other patient suffered from a complication due to this technic of parenteral nutrition. One patient has been treated for 3 months of which 1 month at home. Another one is still on total parenteral nutrition 9 months later with 8 months at home without any complication.

Control of serum protein production in hepatocyte hybridomas: immortalization and expression of normal hepatocyte genes.

"Hepatocyte hybridomas" have been isolated after fusion of adult hepatocytes and alpha-fetoprotein (AFP)-producing mouse hepatoma cells. The yield of viable hybrid clones was low but could be increased by culturing the cells in the presence of insulin. On the basis of their chromosome constitution, the hybrids were classified into two groups characterized by either a single or a double set of mouse (hepatoma) chromosomes. The hybrids segregated rat chromosomes and thus constitute an excellent material for gene mapping studies in the rat. Most of the hepatocyte hybridomas retained the production of one or more rat serum proteins, indicating that the corresponding structural genes, contributed by the normal hepatocyte parent, have been immortalized and maintained in the active state after fusion. However, these hybrids do not produce rat AFP, although mouse AFP synthesis is maintained. This result strongly suggests that silent rat (hepatocyte) AFP genes coexist in hepatocyte hybridoma nuclei with active mouse (hepatoma) AFP genes. Finally, on the basis of certain properties of these hepatocyte-hepatoma hybrids, we suggest that the nondividing state of the hepatocytes is actively controlled by a regulatory mechanism which prevents DNA synthesis or entry into mitosis or both.

Separation in distinct subpopulations by elutriation of liver cells following exposure of rats to N-nitrosomorpholine.

N-Nitrosomorpholine, administered with drinking water to SD rats at the daily dose of 2.4 mg/kg for 7 weeks, induces persisting changes in the hepatocytes as shown by electron microscopy and cytochemistry. In situ, the hepatocytes exhibit a heterogeneous reaction for glucose-6-phosphatase activity. Cells of large diameter, frequently deficient in this enzyme, contain a well-developed rough and/or smooth endoplasmic reticulum. Adult rat hepatocytes from control and N-nitrosomorpholine-treated rats were isolated by enzymatic perfusion. Isolated cell populations in both experimental models were composed of a few contaminating sinusoidal cells; small, intermediate, and large hepatocytes; and doublets or triplets of undissociated cells. Five distinct hepatic subpopulations were separated by elutraition or counterflow centrifugation and analyzed by morphological, morphometric, and cytophotometric methods. Fraction I is composed of small (16 to 18 micrometers) diploid hepatocytes; Fractions II and III consist of homogeneous populations of tetraploid cells (mean diameters, 20.5 and 22.4 micrometers); Fraction IV is enriched with large octoploid cells whose mean diameters reach 25.2 micrometers; and Fraction V contains large cells and cell aggregates. The counterflow centrifugation shows the higher proportion of hypertrophied and polyploid hepatocytes, obtained after carcinogen treatment, in the elutriated Fractions IV and V. The structural integrity of hepatocytes is not affected by the process of elutriation. Large hepatocytes, up to 30 micrometers in diameter, exhibit an abundant smooth endoplasmic reticulum, frequently disposed at the periphery of the cell where it forms a network of anastomosing tubules. Moreover, some of these cells present well-developed rough endoplasmic cisternae, closely associated in large fields. Under the scanning electron microscope, elutriated hepatocytes from control rats show numerous regularly distributed microvilli covering the entire cell surface, whereas hypertrophic hepatocytes from N-nitrosomorpholine-treated rats offer heterogeneous cell surfaces, characterized by the presence of patches of short, closely packed microvilli.

SEM study of adult rat hepatocytes from preneoplastic and hyperplastic foci induced in the liver by diethylnitrosamine.

Oral administration of diethylnitrosamine (DENA) at the final concentration of 70 mg/kg, 24h after partial hepatectomy induces the appearance of preneoplastic foci composed of structurally altered and enzymatic deficient liver parenchymal cells of large size. These lesions grow up and form hyperplastic nodules, 0.5 to 2mm in diameter, 9 to 10 months after DENA treatment. Adult rat hepatocytes were isolated by collagenase perfusion from treated rats, sacrificed 6 months after the carcinogen application. They were separated by elutriation in distinct subpopulations, according to cell size and degree of ploidy. Fractions elutriated at high flow rates are enriched in preneoplastic hepatocytes of large size, which display a heterogeneous distribution of microvilli. Hyperplastic nodules were examined in situ by scanning electron microscopy, in 1 to 2 mm thick tissue slices, 10 months after the carcinogen administration. The nodules, easily detected at this stage of transformation, are composed of hypertrophied hepatocytes which exhibit heterogeneous cell surfaces. The parenchymal cells reveal frequently reduced interhepatocytic faces provided with sinuous bile canaliculi and sinusoidal faces covered with closely apposed, short microvilli.

Synthesis of secretory component by rat hepatocytes in culture.

Normal rat hepatocytes were isolated and cultivated in vitro. Synthesis of secretory component was demonstrated by its accumulation in the culture medium, as measured by radioimmunoassay; by incorporation of 14C-leucine in the protein specifically precipitated with anti-secretory component antiserum; and by a positive precipitin reaction of concentrated culture medium with the same antiserum. The results explain the high levels of secretory component found in rat bile and render plausible a mechanism of hepatic IgA transfer involving secretory component as the hepatocyte membrane receptor for polymeric IgA.

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.