Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line.

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.

Immunocytochemical detection of the onco-developmental protein oncomodulin in pre-neoplastic and neoplastic hepatocellular lesions during hepatocarcinogenesis in rats.

Oncomodulin is a calcium-binding protein, detectable in extra-embryonic human and rat placental cells and in a wide variety of tumors, but not in any normal embryonic or adult rodent or human tissues. It is also absent from proliferatively active fetal or regenerating adult rat liver. The presence of this oncodevelopmental marker was investigated in pre-neoplastic and neoplastic liver lesions during hepatocarcinogenesis induced in rats by DENA treatment, using an antibody raised against purified oncomodulin. Positive immunostaining was observed in foci of altered hepatocytes, in neoplastic nodules and in HC, but not in the histologically normal surrounding liver parenchyma. The proportion of oncomodulin-positive foci gradually rose from 20-25% at 2-3 months after DENA treatment, to about 88% at 6 months and later. The proportion of positive neoplastic nodules increased from 50% at 5 months to about 73% (range 36-100) at 9 months and later; 88% of the HC found 10 to 20 months after DENA treatment were also positive. That early neoplastic nodules are oncomodulin-positive in a proportion (50%) similar to that of foci after the same duration of treatment is consistent with a lineage relationship between them but makes it unlikely that oncomodulin expression conditions the focus-nodule transition. The role, if any, of oncomodulin in malignant progression remains to be elucidated. It seems out of the question that it is a simple correlate of proliferative activity.

Invasiveness, proliferative activity and ultrastructural phenotypes of hepatocytes from diethylnitrosamine-induced neoplastic nodules and hepacarcinomas in vitro.

The invasive behavior of hepatocytes from diethylnitrosamine (DENA) induced neoplastic nodules and hepatocarcinomas was studied in a confronting culture system in vitro. These observations confirm our previous report, demonstrating that hepatocytes from hepatocarcinomas and from neoplastic nodules invaded into embryonic chick precultured heart fragments (PHF), a property associated with malignancy (Mareel, 1979). We now further demonstrate that: (1) Invasiveness was expressed by the hepatocytes in 10 out of 12 samples from hepatocarcinomas, and in 13 out of 36 confronted nodular samples. The hepatocytes from the other two-thirds of nodule samples died off in the confrontation as did all normal hepatocytes. (2) Invasive hepatocytes from tumors and from nodules showed the same arrangement of invasive liver cells in relation to the heart tissue, and the same ultrastructural phenotypes. The latter did not differ from those in the non-invading subpopulations, with the exception perhaps of a higher proportion of cells with an indented nucleus. (3) None of the scored ultrastructural alterations was present in all the invasive cells, thus excluding any specific requisite in this respect. (4) When 3H-TdR was made continuously available to the cultures, starting at the time of confrontation between heart and liver tissues, unlabelled as well as labelled invasive hepatocytes were found inside the PHF in about equal proportions. It is concluded that nodular hepatocytes deviate from normality by at least 2 different properties that may or may not be related to each other and are revealed under in vitro conditions, namely the ability to survive and to proceed through S-phase and mitosis under such conditions and to actively invade precultured chick heart fragments. The latter property indicates that at least some of the nodules contain hepatocytes that have performed one step in malignant progression.

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.

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.

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.

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes.

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.

Ultrastructural and metabolic studies of isolated and cultured hepatocytes.

Cells isolated from adult rat liver form distinct cell populations: the parenchymal cells or hepatocytes and the non-parenchymal cells, mainly endothelial and Kupffer cells. These two groups are easy to separate by centrifugation methods, including centrifugal elutriation. The single-cell suspension of hepatocytes seems to be constituted of subfractions which are located roughly in the centrilobular and perilobular regions of the liver lobule and are differentiated so that they form cell lines with distinct metabolic activities. The basic means of characterizing the isolated hepatocytes consists of determining their size distribution, their sedimentation and elutriation properties, and their metabolic activities. Cultures of hepatocytes offer the possibility of prolonging their survival and of studying the differentiation of new bile canaliculi and the reconstitution of biliary polarity in the cytoplasm. The combination of Kupffer cells with hepatocytes improves the culture condition, as a result of elimination of cell debris by phagocytosis by the non-parenchymal cells. The application of the isolation and partition methods to pre-neoplastic liver allows us to classify the cells according to their degree of hyperplasia and opens up a new field of investigation.