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.     

Negative correlation of L-glutamine concentration with proliferation rate in rat hepatomas

The concentration of L-glutamine was determined in freeze-clamped samples of normal liver of adult male fed rats (5.7-6.1 mumol/g) and in transplantable hepatomas of vastly different proliferative rates. The L-glutamine concentration in the slowly growing hepatomas was in the range of the normal liver and it decreased in relation to the increase of hepatoma growth rate, in the most rapidly growing tumors amounting to 12% of that of normal liver. In 24-hour regenerating liver, the glutamine content was slightly reduced (by 17%). In normal rat organs of high cell renewal, such as testis, intestinal mucosa, spleen, and thymus, the L-glutamine concentration was 18 to 46% of that of normal rat liver. The L-glutamine content was similar in rat brain and liver, but it was 1.6-fold higher in the heart, and low in the blood. Glutamine synthetase (EC 6.3. 1.3) activity in normal adult liver of ACI/N strain rats was 1,000 nmol per hr per mg protein; the activity increased in the very slowly growing hepatoma 20, but decreased markedly in all the other hepatomas. Thus, glutamine synthetase activity was essentially transformation-linked. The negative correlation of glutamine content with growth rate in transplanted hepatomas appears to be more closely linked with the activities of enzymes that utilize glutamine. The low L-glutamine concentration in the rapidly growing hepatomas provides a potential marker for anti-glutamine chemotherapy selectively targeted against the glutamine-utilizing enzymes.     

Hexokinases and glycolysis in embryonic and denervated rat liver]

Certain similarity in the isoenzymic composition of hexokinase (HK) and the rate of glycolysis in embryonic and denervated liver of rats has been revealed. These tissues exhibit high activity of HK-II and especially HK-III, which has the highest affinity to glucose. Depending on the isoenzymic composition of HK, the rate of glycolysis in embryonic and denervated liver is rather intensive even at low external concentrations of glucose and is not significantly affected by the increase in the level of the latter. It is suggested that the observed similarity results from a reduced activity of the sympathetic nervous system. This conclusion is confirmed by low catecholamine content in the tissues studied.     

Electron paramagnetic resonance studies of iron-sulfur centers in mitochondria prepared from three Morris hepatomas with different growth rates

Iron-sulfur centers in mitochondria prepared from Morris hepatomas with different growth rates were compared with those in host liver and nontumor-bearing rat liver mitochondria by EPR measurements (< 77° K). In the slow growing hepatoma 16, EPR signals from iron-sulfur centers located in the NADH dehydrogenase region were specifically diminished. In the rapidly growing hepatoma 7777, EPR signals of all the iron-sulfur centers showed considerably diminished intensity. In hepatoma 7800 having an intermediate growth rate, all iron-sulfur centers showed no change. Those changes in iron-sulfur centers correlated with observed respiratory activities of Morris hepatoma mitochondria. No general correlation was obtained between these parameters and the growth rate of the tumors.     

dUTP pyrophosphatase and uracil-DNA glycosylase in rat liver and hepatomas.

1. The activity of dUTP pyrophosphatase (dUTPase) was similar in rat liver and hepatomas of slow or moderate growth rate but was increased several fold in three rapidly growing hepatomas. 2. There was an approx three-fold increase in the activity of uracil-DNA glycosylase in Morris hepatoma 7800 but there was little change in activity in other hepatomas that were examined. 3. The activities of dUTPase and uracil-DNA glycosylase were not significantly affected by two diets that may be promotional for hepatocarcinogenesis, a high orotate diet and an arginine-deficient diet.     

Activities of some enzymes concerned with citrate and glucose metabolism in transplanted rat hepatomas

1. Certain enzymes concerned with citrate and glucose metabolism have been measured in two transplanted rat hepatomas, one induced with ethionine (minimal deviation type) and one induced with dimethylaminoazobenzene. In these hepatomas both citrate-cleavage enzyme and NADP-linked isocitrate dehydrogenase in the soluble fraction of the cell were approximately one-third of the values for normal rat liver. These changes have been discussed in relation to the increased citric acid content of tumours and depressed rate of fatty acid synthesis. 2. The glucose-ATP-phosphotransferase activity was below normal liver values in the ethionine-induced tumour but greater than normal in the dimethylaminoazobenzene-induced hepatoma. The apparent K(m) values for the glucose-ATP phosphotransferases of these hepatomas were approx. 8x10(-5)m; no evidence was found for an enzyme with a high K(m) for glucose equivalent to liver glucokinase. 3. Of the enzymes of the pentose phosphate pathway, glucose 6-phosphate-dehydrogenase activity was three to five times as great whereas 6-phosphogluconate-dehydrogenase activity was the same or lower than normal liver in the ethionine-and dimethylaminoazobenzene-induced tumours respectively.     

Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 9633 and 7777, and compared to the activities found in normal and host liver. A 2-3-fold increase was found in the phosphatidylcholine and phosphatidylinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.     

Multiple forms of uridine kinase in normal and neoplastic rat liver

Two species of uridine kinase with molecular weights of approximately 120000 (I) and 30000 (II) have been identified in the rat liver system. Species I predominates in the 7-day postnatal and adult rat liver and increases in the regenerating remnant of the latter after partial hepatectomy; the concentration of species II is low in these tissues. Species I also predominates in the slow-growing hepatomas 5123D and 7800. In contrast, II is the predominant form in the foetal rat liver and accounts for 40% of the total activity in the rapidly growing Novikoff ascites hepatoma. In contrast to species II, which was stable, species I was inactivated by preincubation for 30min at 37 degrees C, before assay at 23 degrees C.     

Carbohydrate metabolism enzymatic activity and its alteration under the influence of thyroid hormone during tumor growth]

The activity of hexokinase (HK), its isoenzymes, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase, and the triiodothyronine (T3) effect on this activity in the liver tissue of mice bearing transplantable hepatoma 22a were studied in different periods of the tumor growtn. It was shown that alterations in the activity of the enzymes in the liver of tumor-bearing mice occurred already in the presence of a small tumor. More profound alterations in the activity of all enzymes studied, apart from those in the enzymatic pattern of HK, could be observed starting from day 21after the tumor transplantation. In the initial stages of the hepatoma growth the activity of the test enzymes in the liver was regulated by thyroid hormone. The effect of Ta on the activity of the enzymes in the host liver was gradually lost in the course of the tumor growth.     

Regulation of lipoprotein receptors on rat hepatomas in vivo

It has been shown previously that the rat hepatoma no. 7288C grown in vivo or in vitro expresses fewer receptors which recognize chylomicron remnants than does normal rat liver, and it was suggested that this may contribute to the deletion of dietary cholesterol-induced regulation of cholesterol synthesis in hepatomas (Barnard, G., Erickson, S. and Cooper, A. (1984) J. Clin. Invest. 74, 173-184). To investigate this further, Buffalo rats bearing hepatomas (HTC no. 7288C) were made hypercholesterolemic by feeding an atherogenic diet and hypocholesterolemic by ethinyl estradiol injections. Under all circumstances, tumor membranes had fewer receptors than liver membranes as measured by specific binding of [125I]chylomicron remnants. Ethinyl estradiol treatment increased the number of lipoprotein receptors 1.7-fold in liver membranes and 1.2-1.6-fold in tumor membranes, but hypercholesterolemia did not produce any significant changes in remnant binding to either liver or hepatoma membranes. Feeding an atherogenic diet induced a 2.4-fold increase in total cholesterol content in the liver, primarily as cholesterol ester; however, there was no change in total, free or ester cholesterol in the hepatomas. Acyl coenzyme A:cholesterol acyltransferase activity was low in this hepatoma line and neither treatment significantly affected its activity. One explanation for the lack of effect of the atherogenic diet on hepatoma cholesterol metabolism in addition to the decreased number of lipoprotein receptors might be the failure of access of lipoproteins to the tumor cell. To assess this, radioiodinated apo E-rich lipoproteins of various sizes were injected intravenously into rats with hepatomas. Their disappearance from the circulation was followed, and the uptake of each lipoprotein into a variety of tissues was determined. Chylomicron remnants were the most avidly removed particles. VLDLH, IDLH and HDLC were removed more slowly and less completely. None of the lipoproteins accumulated substantially in the tumors suggesting a limited access to the hepatoma tissue. Thus, in addition to the observed reduction in lipoprotein receptor number, limited lipoprotein access to the hepatoma tissue may be a significant factor in contributing to the apparent lack of feedback regulation of cholesterol synthesis by hepatoma tissue in vivo.     

Hepatic glycogen synthesis in the absence of glucokinase: the case of embryonic liver

Glucokinase (GK, hexokinase type IV) is required for the accumulation of glycogen in adult liver and hepatoma cells. Paradoxically, mammalian embryonic livers store glycogen successfully in the absence of GK. Here we address how mammalian embryonic livers, but not adult livers or hepatoma cells, manage to accumulate glycogen in the absence of this enzyme. Hexokinase type I or II (HKI, HKII) substitutes for GK in hepatomas and in embryonic livers. We engineered FTO2B cells, a hepatoma cell line in which GK is not expressed, to unveil the modifications required to allow them to accumulate glycogen. In the light of these results, we then examined glycogen metabolism in embryonic liver. Glycogen accumulation in FTO2B cells can be triggered through elevated expression of HKI or either of the protein phosphatase 1 regulatory subunits, namely PTG or G L. Between these two strategies to activate glycogen deposition in the absence of GK, embryonic livers choose to express massive levels of HKI and HKII. We conclude that although the GK/liver glycogen synthase tandem is ideally suited to store glycogen in liver when blood glucose is high, the substitution of HKI for GK in embryonic livers allows the HKI/liver glycogen synthase tandem to make glycogen independently of the glucose concentration in blood, although it requires huge levels of HK. Moreover, the physiological consequence of the HK isoform switch is that the embryonic liver safeguards its glycogen deposits, required as the main source of energy at birth, from maternal starvation.     

Acquirement of fetal properties in hepatoma on glutathione metabolism

Glutathione peroxidase/glutathionè reductase activity ratio was determined in the high-speed supernatant fraction of the rat livers. The ratio was dependent on age and the ratio increase gradually with the increase in age. The fetal liver showed a ratio of 1.5–2.0, which was almost the same value to those of the 4-dimethylaminoazobenzene-induced primary hepatoma and some transplantable hepatomas originating from the azodye-induced hepatoma. Four cell lines of transplantable ascites hepatoma examined in this study showed the value of 1.2–1.8 for the activity ratio, however, the values of two strains were found to be 2.8–3.0, even though these cell lines were also originated from the azodye-induced hepatoma.Glutathione contents of azodye-induced hepatoma and ascites hepatomas were also similar with those of fetal rat livers.The acquirement of the fetal properties in hepatoma was discussed in relation to glutathione metabolism.     

Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma.

The subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase is altered in the Morris 7777 hepatoma. Mitochondria in this poorly differentiated tumor are the principal sites of CDP-diacylglycerol synthesis, in contrast to normal rat liver where the endoplasmic reticulum is most active. This enzyme activity was increased 17-fold in the outer mitochondrial membrane, and a 22% increase was noted in the inner mitochondrial membrane of the 7777 hepatoma as compared with the corresponding fractions from normal rat liver. Increased mitochondrial CTP:phosphatidate cytidylyltransferase was present in six other Morris hepatomas, but it was not found in fetal rat liver mitochondria, suggesting that rapid growth alone is not responsible for the difference. Evidence is presented which indicates that mitochondrial lipid degradation is similar in normal liver and the 7777 hepatoma, in vitro. The increased activity of CTP: phosphatidate cytidylytransferase is thought to be responsible in part for the moderately increased diphosphatidylglycerol content of 7777 hepatoma mitochondria.     

Isoenzymes of blood serum alkaline phosphatase under experimental cholemia

The activity and isoenzymic spectrum of alkaline phosphatase of the blood serum and liver under cholemia caused by deoxycholic acid were compared in healthy animals and in animals with the affected liver. It is shown, that under conditions of the bile acids higher content in the organism due to deoxycholic acid, the total activity increases considerably and there appears an isoenzyme absent in the blood serum of healthy animals. Changes in the activity and isoenzymic spectrum of alkaline phosphatase under experimental cholemia developing against a background of the healthy and affected liver are characterized by certain peculiarities.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

Isolation and characterization of the plasma membranes from rat ascites hepatomas and from normal rat livers, including newborn, regenerating, and adult livers.

Plasma membranes (PM) were isolated from island-forming types of rat ascites hepatoma (AH 130, AH 602, and AH 7974) and from their free-cell sublines (AH 130FN and AH 7974F), and were characterized in terms of electron-microscopic morphology, marker enzyme activities, and lipid contents. The results were compared with those of the PM isolated in a similar way from newborn, regenerating, and adult livers. The marker enzyme activities, such as Na+, K+-insensitive Mg2+-ATPase [EC 3.6.1.3] (Mg2+-ATPase) and 5'-nucleotidase [EC 3.1.3.5], as well as the phospholipid composition of the PM isolated from hepatomas by Wallach's nitrogen gas cavitation method were similar to those obtained with the PM isolated by a modification of Emmelot's method, although the former method gave a much lower yield in terms of protein than the latter. Based on the modified Emmelot method, sufficiently pure PM preparations could be obtained from the hepatomas in the form of large membrane sheets without any contamination by other identifiable components, as determined with an electron microscope, and with high specific activities of the marker enzymes, such as Na+, K+-sensitive ATPase [EC 3.6.1.3] (Na+, K+ -ATPase), Mg2+ -ATPase, and 5'-nucleotidase. As for the characteristics of the hepatoma PM, lower specific activity of 5'-nucleotidase and higher fatty aldehyde molar percentages in total phospholipids were noted in all the PM from the hepatomas in comparison with normal liver PM of various origins. The PM from the hepatomas showed an increased amount of cholesterol (mumole per mg protein), whereas actively growing newborn and regenerating livers gave rather lower amounts in comparison with that of normal adult liver.     

Aldehyde dehydrogenase in 2-acetaminofluorene-induced rat hepatomas. Ontogeny and evidence that the new isoenzymes are not due to normal gene de-repression

The pre- and post-natal ontogeny of Sprague-Dawley rat liver aldehyde dehydrogenase [aldehyde-NAD(P)(+) oxidoreductase, EC 1.2.1.5] is described. At no time in its ontogenetic development does normal liver aldehyde dehydrogenase exhibit any of the characteristics of a series of unique aldehyde dehydrogenases that can be isolated from 2-acetamidofluorene-induced rat hepatomas. Enzyme activity is first detectable in 15-day foetal liver and gradually increases throughout pre- and post-natal development until adult activities are attained by day 49 after birth. Electrophoretically, normal aldehyde dehydrogenase, throughout its ontogeny, exists as the same single isoenzyme found in normal adult liver. Isoelectric points for two normal liver isoenzymes demonstrable by isoelectric focusing are pH5.9 and 6.0. The immunochemical properties of aldehyde dehydrogenase during its ontogeny are identical with those of normal adult liver aldehyde dehydrogenase when tested against anti-(hepatoma aldehyde dehydrogenase) serum in Ouchterlony double-diffusion tests. The results indicate that the hepatoma-specific aldehyde dehydrogenases are not the result of the de-repression of genes normally repressed in adult rat liver or in some other adult tissue.     

Base composition studies on mitochondrial 4 S RNA from rat liver and Morris hepatomas 5123D and 7777.

The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.     

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.     

Ornithine decarboxylase activity and DNA synthesis in Morris hepatomas 5123-C and 7800.

The activities of ornithine decarboxylase (ODC) and thymidine kinase (TK) and the rates of DNA synthesis were determined in hepatomas and livers of rats bearing Morris hepatoma 5123-C or 7800 and entrained to a schedule of 12 hours of light followed by 12 hours of darkness, with food (60% protein) available only during the first 2 hours of the dark period. ODC activity in hepatoma 5123-C displayed a diurnal oscillation, increasing 2-fold during the feeding period and then rapidly decaying to 20% of the peak level. The livers of rats bearing hepatoma 5123-C exhibited a similar oscillation of ODC activity, with peak values lower than in the hepatomas but higher than in the livers of control (non-tumor bearing) animals. TK activity and the rate of DNA synthesis in hepatoma 5123-C were low during most of the dark period but increased rapidly towards the end of the dark period. DNA synthesis reached a plateau at the dark-light interface and then rapidly declined, but TK activity remained high during the light period. Similar studies on hepatoma 7800 established that ODC activity in this hepatoma did not oscillate but remained at low levels throughout the day. Similarly, host livers of rats bearing hepatoma 7800 did not exhibit the diurnal oscillation of ODC activity characteristic of liver from control rats, but showed a slow increase in activity followed by a plateau and a slow decline to base-line levels. DNA synthesis in hepatoma 7800 was constant throughout the day, whereas TK activity may have increased during the dark period. In the livers of control rats and animals bearing hepatoma 5123-C or 7800, TK activity and rate of DNA synthesis were at low levels at all times studied and appeared not to oscillate.