RNAs containing mitochondrial ND6 and COI sequences present an abnormal structure in chemically induced rat hepatomas.

We have constructed a cDNA library prepared from an hepatoma cell line (HTC cells) and isolated a clone, pHT 13, which corresponds to mRNAs present at a much higher level in rat hepatomas than in normal hepatocytes. The sequence of the pHT 13 insert has been previously published (Nucleic Acids Res. 1988, 16,10935). This clone contains mitochondrial DNA sequences with an abnormal organization, since it includes part of the NADH dehydrogenase subunit 6 (ND6) and of the cytochrome oxidase subunit I (COI) genes separated by 230 bases instead of 9 kb in mitochondrial genome from normal hepatocytes. In this work we show (1) that RNAs homologous to this clone are present in hepatoma cells but not in normal hepatocytes, (2) that a 3 kb fragment of tumor mitochondrial DNA contains both the ND6 and the COI sequences. The abnormal structure of the DNA is confirmed by Southern blot analysis which shows that distinct types of mitochondrial DNAs are present in hepatoma cells.     

Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes.

The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of chromosomal nonhistone proteins pp23 in cells from different types of tissue]

A specific immunoserum against nonhistone chromosomal protein pp23 from rat kidney was produced. A similar protein was obtained in cells of different tissue specificity (in the liver cells of rats submitted to a single effect of hepatocarcinogens, in rat hepatomas G-27, HTC, Zajdela, in cultivated non-tumor myogenic L6 cells and in kidney cells of newborn rats) by means of immunofluorescent method on using this immunoserum. In all cases a specific fluorescence of the nucleus and nuclear membrane was observed.     

Prostaglandin biosynthetic capacity of hepatomas with different growth rates

1. Prostaglandin synthesis from [14C]arachidonate by microsomal fractions was measured with preparations from rat liver and from hepatomas of different growth rates. The highest rates of synthesis were observed with microsomal preparations from the rapidly growing hepatoma HTC. 2. Assay of endogenous levels of prostaglandins E2, F2 alpha and thromboxane B2 also indicated high levels in solid tumors of the HTC line. 3. With HTC cells in culture it was necessary to incubate in the absence of serum in order to detect prostaglandin synthesis. 4. The data indicated that, while prostaglandin synthesis was elevated in HTC cells, the synthesis of prostaglandins by a series of hepatomas was not closely correlated with the growth rates of the tumors.     

Immunomodulating effect of cyclophosphamide on cytotoxic activity of rats and mice splenocytes

This study examines the immunomodulating effect of cytostatic drug--cyclophosphamide (Cy)--on natural cytotoxic activity of rats and mice splenocytes. The cytotoxicity of the effector cells against the confluent monolayer cell lines of rat hepatoma Zajdela and HTC and mice hepatoma MH-22a was estimated by means the morphometric analysis. It was shown, that 48 h after single intraperitoneal injection of Cy produced a immunomodulating effect on the activity of splenocytes--suppressor action on cells of Zajdela hepatoma and immunopotentiating action on the target cells of HTC and MH-22a hepatomas. The possible mechanisms of immunopotentiating effect of Cy are discussed.     

Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

The effect of preparations of non-histone chromosomal proteins on the expression of hetero-organic membrane antigens of kidney origin in a primary rat hepatocyte culture

The expression of membrane hetero-organic antigens of kidney origin, associated with the rat hepatomas in primary culture of intact adult rat hepatocytes, was investigated by means of the indirect immunofluorescence method using a specific immune serum. These antigens were observed on the membrane of some hepatocytes after their contact with nonhistone chromosomal proteins (NHCP), which were obtained from the kidney of intact rats from cells of hepatoma 27 and Zajdela hepatoma, or from the carcinogenic liver after a single diethylnitrosamine injection. Negative results were obtained after the incubation of hepatocytes in the medium lacking some of NHCP, or in that with NHCP obtained from the liver of intact rats.     

Expression of the bile acid transport protein during liver development and in hepatoma cells

The expression of the hepatocyte Na(+)-dependent bile acid transport protein during liver development and in hepatoma cells has been characterized using a monoclonal antibody (mAb 25D-1) which specifically recognizes this 49-kDa carrier system. mAb binding studies demonstrated a greatly reduced concentration of this transport protein on the surface of hepatoma tissue culture (HTC) cells, a result consistent with the greater than 95% reduction in bile acid transport capacity when compared with normal adult hepatocytes. Immunoprecipitation procedures with 25D-1 were utilized to quantitate the presence of this transport protein in HTC cells as well as in adult hepatocytes that had been labeled with [35S]methionine or Na125I. These studies indicate that the 49-kDa transport protein is not expressed either on the surface or in any intracellular compartment in HTC cells. mAb binding to fetal cells (day 17) also indicated a greatly decreased number of transport molecules in the plasma membrane. Total cell content of this carrier protein during the next 7 weeks of liver development, as measured by immunoprecipitation, increased in a linear fashion reaching 92% of the adult level at 4 weeks after birth, which parallels the increase in transport function. These results demonstrate that bile acid transport capacity is directly related to the level of expression of this 49-kDa membrane protein.     

The effect of adenosine and chloroadenosine on sex differences in the energy metabolism of rat hepatocytes

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).     

Expression of hepatic mitochondrial carbonic anhydrase V

We have raised specific (rabbit anti-rat) polyclonal antibodies to hepatic mitochondrial carbonic anhydrase V (CA V) and used them to assay the amounts of protein expressed in liver mitochondria isolated from term-foetal, control or diabetic adult rats and in perivenous and periportal rat hepatocytes. The levels of CA V expressed in mitochondria isolated from the livers of adult male and female rats are similar and increase (about 2-fold) in mitochondria from adult diabetic rats when compared to those isolated from the livers of control rats. The level of enzyme in adult liver was higher than in the livers of term-foetal rats. CA V is expressed in both perivenous and periportal hepatocytes, but the level of expression is greater (approx. 40%) in perivenous cells. The implications and significance of these findings are discussed with reference to the roles and properties of the other carbonic anhydrase isoenzymes and the metabolic function of the mitochondrial isoenzyme.     

Dexamethasone regulates the program of secretory glycoprotein synthesis in hepatoma tissue culture cells

The secretory glycoproteins synthesized by hepatoma tissue culture (HTC) cells were resolved by two-dimensional polyacrylamide gel electrophoresis of media from cells that were grown in the presence of [(3)H]fucose. These cells synthesize and secrete a complex set of fucose-containing glycoproteins. These secretory glycoproteins are distinct from those glycoproteins present in the plasma membrane of HTC cells. Incubation of HTC cells with dexamethasone has a pronounced effect on the quality and quantity (denoted here as the program) of secretory protein synthesis, as assayed by the short-term incorporation of labeled mannose, fucose, or methionine. The synthesis of two mannose- and fucose- containing glycoprotein series, one of 50,000 mol wt and a more heterogeneous series with mol wt of 35,000-50,000, is increased to a high level by the hormone; conversely, the synthesis of other secretory proteins, particularly one with mol wt of 70,000, is decreased or stopped completely. The synthesis of some major secretory proteins is not affected by the hormone. Dexamethasone has less of an effect on the composition of either total cell membrane glycoprotein or plasma membrane glycoprotein. But there is a decrease in the synthesis of a major membrane glycoprotein series with mol wt of 140,000. These effects of dexamethasone are relatively specific to HTC cells. Neither Reuber H-35 cells nor primary cultures of rat hepatocytes show the same response to the steroid. Two variant HTC cell lines, which were selected for their resistance to dexamethasone inhibition of extracellular plasminogen activator activity, respond only partially to the steroid-induced regulation of the secretory and membrane glycoproteins.     

N-Nitrosomorpholine induced alterations of unscheduled DNA synthesis, mitochondrial DNA synthesis and cell proliferation in different cell types of liver, kidney, and urogenital organs in the rat

In order to measure rates of unscheduled DNA synthesis (UDS), mitochondrial DNA synthesis, and cell proliferation, i.e. factors relevant in the early phase of carcinogenesis, young rats received by gavage 200 mg/kg N-nitrosomorpholine (NNM) or vehicle (distilled water), and were injected with 3H-thymidine 24 h later. Autoradiographs from liver, kidney, urethra, prostate, seminal vesicle, and ductus deferens were prepared from deparaffinized sections, using a 250-day exposure time. In the liver, UDS was at least doubled in 2n and 4n hepatocytes. Approximately 3% of these hepatocytes exhibited a fourfold increase in UDS. Such strongly labeled cells were only observed in the liver following NNM exposure. With the exception of renal epithelial cells of the proximal tubule, UDS in epithelial cells of bladder, urethra, ductus deferens, seminal vesicle and prostate was decreased in NNM-exposed rats. Mitochondrial DNA synthesis and cell proliferation were significantly increased only in hepatocytes, and were decreased in all other monitored organs in NNM-exposed rats. The strongly increased UDS and more moderately increased mitochondrial DNA synthesis in a subgroup of hepatocytes suggest that possibly some unrepaired damage persists in the DNA of these cells. The latter cells may be the precursors of so-called foci of hepatocellular alteration, which appear later during the process of carcinogenesis. The increased UDS but decreased rate of proliferation in the renal proximal tubule cells might be related to renal carcinogenesis which is observed in NNM-exposed rats after a long latency period.     

Biochemical characteristics determining the rates of tumour growth in the organism]

Experimental data suggest that contrary to the findings obtained for normal and regenerating liver of mouse, the greater part of hexokinase (HK) in transplantable hepatomas is firmly bound to mitochondrial membranes. It is shown that the ratio of the bound HK activity (HKbound) to that of total HK activity (HKtotal) diminishes with a hepatoma growth. Malignization of hepatocytes also leads to a sharp decrease in the cytochrome oxidase (CO) octivity. Though the data obtained are well-correlated with the Warburg hypothesis, there is no direct correlation between the malignancy of hepatomas evaluated by their growth rates, and the biochemical parameters of the tumours studied. On the basis of fundamental principles of Warburg's, it is proposed to evaluate energy metabolism of hepatomas by the activity and subcellular distribution patterns of HK as well as be the activity of CO, according to the expression: [(HKtotal)2//HKbound-CO+HKbound-CO]. It is demonstrated that there exists a certain linear dependence between the integral characteristics of hepatoma energetics and their growth rates.     

Uptake and metabolism of eicosa-8,11,14-trienoic acid in normal hepatocytes and HTC cells

Summary The incorporation and conversion of eicosa-8, 11, 14-trienoic acid to arachidonic acid were studied in HTC cells (7288 c hepatoma cells) and isolated hepatocytes of rat. The cells were incubated at different concentrations of the acid during 0 to 6 hours. Both kinds of cells were able to take up the acid. However, the HTC cells have a greater avidity for the eicosatrienoic acid than normal liver cells. The incorporation of the acid modified the fatty acid composition of the cells and caused a decrease in the amount of saturated and monoenoic acid. In both cells eicosatrienoic acid was converted to arachidonic acid. However, in HTC cells arachidonic acid level was low and not correlated with the amount of the eicosatrienoic acid incorporated. This fact is apparently not due to an impairment in 5 desaturation activity since this enzyme is active in both cells. It would be possible that arachidonic acid production in malignant cells would be also interrupted in another metabolic pathway after 6 desaturation step. The strikingly low amount of arachidonic acid biosynthesized in HTC cells compared to normal hepatocytes could be interpreted as a consequence of a lower availability of eicosatrienoic acid for the microsomal desaturation system in malignant cells, in addition to the low A6 desaturase activity.     

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.     

The protein kinases tightly bound to DNA are present in normal tissues and in regenerating liver, but strongly decreased in hepatomas

We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation.     

Morphometry of hepatocyte mitochondrial apparatus in normal and cirrhotic rat liver

Morphometric electron microscopy study of the hepatocyte mitochondrial apparatus and morphofunctional analysis of the degree of pathological alterations were carried out on the liver of rats with CCL4-cirrhosis (experimental group). Chronic poisoning of rats with CCL4 for 6 months led to a 4.2-fold increase in proportion of connective tissue and to a decrease in the number of hepatocytes in the liver by 21.8 %. Dry mass and ploidy of hepatocytes in the cirrhotis liver rose as compared with norm by 20.6 and 9.3%, respectively. Activities of alanine and aspartate aminotransferases in blood of rats of experimental group exceeded normal ones 2.0 and 1.4 times, respectively. Concentration of total bilirubin in blood of the cirrhotic animals increased 1.7 times, while concentration of total protein decreased by 22%. Concentration of diene conjugates in the liver of rats of experimental group increased 2.1 times as compared with normal one, while the level of malonic dialdehyde - by 34%. Activities of superoxide dismutase and catalase in the cirrhotic liver were lower than in the normal liver were lower than in the normal liver by 16 and 23 %, respectively. Morphometry of the hepatocyte mitochondrial apparatus has shown that in spite of an increase in the voluminous density of mitochondria in hepatocytes of the cirrhotic liver (by 28 %), concentration of internal mitochondrial membranes in the cells was reduced almost 1.5 times, while the total length of internal membrane in a single mitochondrion was reduced about twice as compared with norm. Thus, despite compensation of the partial loss of hepatocytes because of their polyploidization and hypertrophy, the specific synthetic activity of cells in the case of cirrhosis is decreased due to deterioration of the antioxidant system and electron transport chain of the mitochondrial apparatus.     

REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE AND TYROSINE TRANSAMINASE IN HEPATOMA CELL CULTURES : III. Comparative Studies in H35, HTC, MH1C1, and RLC Cells

The ability of N⁶, O²'-dibutyryl cyclic AMP (DBcAMP) to regulate a number of metabolic events in four lines of cultured rat hepatomas has been examined. Although dexamethasone induces tyrosine transaminase in all four lines, DBcAMP induces this enzyme normally only in H35 cells. A slight increase in transaminase activity was seen with MH₁C₁ cells and HTC cells, but no effect was detectable in RLC cells. In contrast, phosphoenolpyruvate carboxykinase activity is increased by both agents in H35 and MH₁C₁ cells, but neither had any effect in HTC or RLC cells. DBcAMP caused a rapid inhibition of the growth rate and DNA synthesis and an increase in protein content in both H35 and MH₁C₁ cells but not in HTC or RLC cells. The effect of DBcAMP on DNA synthesis in MH₁C₁ cells could be reversed by deoxycytidine as is also the case with H35 cells. The resistance of HTC and RLC cells to DBcAMP was not due to reduced uptake or deacylation as judged by studies with [³H]DBcAMP. The cyclic nucleotide appears to enter the cells by passive diffusion as the intracellular concentration approaches that in the medium within 30–60 min. Possible explanations for the differential responses observed are discussed.