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.     

Colon tumor: enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.     

Role of ribonucleotide reductase in expression of the neoplastic program

Evidence is presented for the tight linkage of ribonucleotide reductase activity with normal and neoplastic proliferation. A sensitive and reproducible assay was worked out to measure CDP reductase activity in rat in normal liver and various tissues, hepatomas of different growth rates, kidney tumors and sarcoma and tissue culture cells of hepatoma 3924A. In the standard assay, linear kinetics were obtained and the reductase activity of the rat liver was 23 ± 3 pmol CDP metabolized per hr/mg protein. When hepatoma 3924A tissue culture cells that had accumulated in plateau phase were replated, allowed to go through lag and log phases and again into the plateau phase during a 96-hr period, ribonucleotide reductase activity rose at 6 hr after cells were plated, the activity was maintained at high levels during the first 48-hr period, and returned to the resting level at 72 and 96 hr. This rise was earlier than that of 6 other enzymes of pyrimidine $\text{de novo}$ and salvage pathways (thymidine kinase, CTP synthetase, orotidine-5′-phosphate decarboxylase, orotate phosphoribosyltransferase, uridine phosphoribosyltransferase, and uridine-cytidine kinase). The rise in reductase activity was synchronous with the increase in incorporation of cytidine and deoxycytidine in the hepatoma cells. The reductase activity was markedly elevated in kidney tumors (31-fold) and in sarcoma (60-fold) as compared to the kidney cortex and muscle, respectively. In 14 lines of transplantable solid hepatomas, reductase activity was increased from 6.2- to 326-fold of that of normal rat liver. The rise in reductase activity positively correlated with the growth rate of the hepatomas; the behavior of CDP reductase was both transformation- and progression-linked. Reductase activity was also high in differentiating and regenerating liver; thus, it also was linked with normal proliferation. However, the elevation in activity was more marked in the rapidly-growing solid hepatoma 3924A (97-fold) than in normal tissues with the same replicative rate, such as regenerating (56-fold) or differentiating (46-fold) liver. Reductase activity was also high in organs of active cell renewal (thymus, bone marrow, spleen and intestine). Since in the solid hepatomas the levels of the substrate for the reductase, the ribonucleoside diphosphates, were generally unaltered, the marked elevation observed in the concentration of deoxynucleoside triphosphates may be attributed primarily to the early and marked rise in CDP reductase activity.     

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.     

Simple separation of tritiated water and [3H]deoxyuridine from [5-3H]deoxyuridine 5'-monophosphate in the thymidylate synthase assay

A simple micromethod was developed for the accurate measurement of the activity of dTMP synthase in rat liver crude extracts. The reaction product of dTMP synthase activity assay, i.e., tritiated water, generated by the release of tritium from carbon-5 of [5-3H]deoxyuridine 5'-monophosphate (dUMP), was separated simply by 100% KOH absorption from [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]deoxyuridine (dUrd), which is the side-product by dephosphorylation of [5-3H]dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while [3H]dUrd remained in the bottom of vessels after absorption of the substrate, [5-3H]dUMP, from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extract of rat liver, the specific activities of dTMP synthase and dUMP phosphatase were 0.092 +/- 0.002 and 0.351 +/- 0.013 nmol/h/mg protein, respectively. This method was also adapted for dTMP synthase assay in crude extracts of rat hepatoma 3924A. The major advantages of this procedure are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity in crude extracts, one-step separation of 3H2O, high sensitivity (with a limit of detection of 10 pmol of 3H2O production), high reproducibility (less than +/- 4.3%), and capability to measure activity in small amounts of sample (30-45 micrograms protein).     

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.     

Partial purification, properties and regulation of inosine 5''phosphate dehydrogenase in normal and malignant rat tissues.

IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5--13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

Biochemical and molecular properties of cisplatin-resistant A2780 cells grown in folinic acid

In this study, A2780 human ovarian carcinoma cells were grown in folinic acid in contrast to folic acid, and the molecular and biochemical properties of cisplatin-resistant A2780 cells were analyzed for changes in the dTMP synthase cycle. At concentrations of folinic acid that were optimal for cell growth (10(-8) M), the ED50 for cisplatin was 2.5 and 43 microM in the A2780S and A2780DDP cells, respectively. Resistance to cisplatin was associated with a 2-fold cross-resistance to 5-fluorodeoxyuridine and 5-fluorouracil as well as a 3-fold increase in both dTMP synthase activity and mRNA. The ED50 for methotrexate was similar in both A2780S and A2780DDP cells (1.2 microM). When both the A2780S and A2780DDP cells were grown in folinic acid, there was no significant difference in the level of dihydrofolate reductase activity. This data would suggest that cisplatin resistance is associated with changes in folate metabolism.     

Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas.

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.     

Poly (adnenosine diphosphoribose) synthase activity of isolated nuclei of normal and leukemic leukocytes.

Poly(adenosine diphosphoribose) (ADPR) synthase activities of nuclei isolated from normal human and leukemic leukocytes were assayed by incubation with radioactive NAD+. The synthase activity of leukemic leukocyte nuclei was significantly higher than that of normal leukocyte nuclei. The average length of polymers formed by isolated leukemic nuclei under the prescribed experimental condition ranged from 3.1 to 5.3 ADPR residues per chain, while those produced by normal leukocytes nuclei was 1.7 and 2.6 residues per chain. Isolated leukemic and normal leukocyte nuclei were incubated with and without NAD+ and the ability to carry out DNA synthesis was measured. The endogenous DNA synthesis of NAD-treated and untreated nuclei was the same. This finding parallels the result obtained with Novikoff hepatoma cell nuclei and differs from the observation with rat liver or testis nuclei.     

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.     

Methotrexate decreases thymidine kinase activity.

MTX cytotoxicity is not fully explained by its well-known inhibition of dihydrofolate reductase activity which leads to a decrease in the dTMP synthase reaction, since TdR kinase which converts TdR to dTMP could readily circumvent MTX action through this salvage activity. TdR kinase is of particular significance, since in various types of carcinoma cells its activity is orders of magnitude higher than that of dTMP synthase. To throw light on this problem, we tested the hypothesis that the impact of MTX treatment might in fact involve an inhibition or decrease in TdR kinase activity. Injection in rat of MTX (i.p.) decreased TdR kinase activity in a time- and dose-dependent fashion in liver (t1/2 = 46 h; IC50 = 95 mg/kg), bone marrow (t1/2 = 10 h; IC50 = 5 mg/kg) and rapidly growing transplantable hepatoma 3924A (t1/2 = 56 h; IC50 = 5 mg/kg). Injection in rat of cycloheximide (15 mg/kg, i.p.), an inhibitor of protein biosynthesis, rapidly decreased TdR kinase activity in the hepatoma (t1/2 = 3.6 h); activities of other purine and pyrimidine synthetic enzymes, dTMP synthase, IMP dehydrogenase, GMP reductase and GMP synthase, declined at a markedly slower rate (t1/2 = 11, 11.6, 12 and 22 h, respectively). MTX, by curtailing purine and pyrimidine biosynthesis, limits product of TdR kinase which is more sensitive to unopposed protein degradation than other enzymes of nucleic acid biosynthesis. TdR kinase is a newly discovered target of MTX treatment.     

Distribution of glutamine hexosephosphate aminotransferase in rat tissues; changes with state of differentiation

The activity of glutamine hexosephosphate aminotransferase (L-glutamine: D-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was determined with an improved assay method in some three dozen rat tissues: adult, developing and neoplastic. The highest activities (20–200 units/g) were seen in colon, mammary (during late lactation), submaxillary, sublingual and parotid glands, placenta and liver. The activity increased strikingly along the length of the intestine; glucose feeding inhibited it in ileum and colon. In liver and intestine the activity increased with age but in brain, muscle, heart and kidney the activity was considerably higher during fetal (7.1–12.8 units/g) than in adult life (0.8–3.5 units/g). Renal, mammary and muscle tumors (but not hepatomas) had much higher activities (4–20.5 units/g) than the cognate normal adult tissue.The distribution pattern among tissues indicates that glutamine hexosephosphate aminotransferase is of general importance to all growing, undifferentiated tissues and of special importance to the differentiated function of particular adult organs. The latter are organs which engage in glycoprotein secretion. The results support the assumption that glutamine hexosephosphate aminotransferase activity is essential for glycoprotein synthesis.     

Nicotinamide methylation tissue distribution,developmental and neoplastic changes

The distribution of cytosolic activity of nicotinamide:S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G₁-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.     

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.     

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.     

Influence of age, sex and cancer on the activities of gamma-glutamyl transpeptidase and of dipeptidyl aminopeptidase IV in rat tissues

gamma-Glutamyl transpeptidase (GTP; EC 2.3.2.2) is an enzyme known to show activity changes during development and carcinogenesis. Its activity was measured in the livers and lungs of female and male rats of different ages, in Morris hepatomas and in experimentally induced pancreatic carcinomas. For comparison purposes, the activity of another peptidase, dipeptidyl aminopeptidase IV (DPAP; EC 3.4.14.1), was assayed in the same tissues. GTP activity was high in fetal liver and hepatomas, but low in adult rat liver, with a marked sex difference, 3 times higher in the female than in the male. In the pancreas, however, the activity of the enzyme was high in the adult but low in the fetus and in pancreatic carcinoma. There were no marked developmental changes or sex differences in pulmonary GTP activities. DPAP levels were low in fetal and neonatal liver and lung, they increased rapidly after birth and showed no sex differences in the adult. In Morris hepatomas and in pancreatic tumors the activity of DPAP was significantly lower than in normal adult liver and pancreas. These results suggest that measurements of GTP (and, to a lesser extent, DPAP) are remarkably suitable for the study of neoplastic cells and tissues.     

Developmental changes in the expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers

Cloned cDNAs encoding a number of enzymes involved in cholesterol biosynthesis as well as extracellular and intracellular lipid transport were used to compare the developmental maturation of these biologic functions in the fetal and neonatal rat and human liver. The results of RNA blot hybridization analyses indicate that steady-state levels of rat HMG-CoA synthase, HMG-CoA reductase and prenyl transferase mRNAs are highest in late fetal life and undergo precipitous (up to 80-fold) co-ordinate reductions immediately after parturition. These changes reflect the ability of the fetal rat liver to produce large quantities of cholesterol as well as the repression of this function during the suckling period in response to exogenous dietary cholesterol. Striking co-ordinate patterns of HMG-CoA synthase, reductase and prenyl-transferase mRNA accumulation were also observed in four extrahepatic rat tissues (brain, lung, intestine and kidney) during the perinatal period. The concentrations of all three mRNAs in the 8-week-old human fetal liver are similar to those observed throughout subsequent intrauterine development with less than 2-fold changes noted between the 8th through 25th weeks of gestation. Analysis of the levels of human apo AI, apo AII, apo B and liver fatty acid binding protein mRNAs during this period and in newborn liver specimens also indicated less than 2-3-fold changes. These observations suggest that the 8-week human liver has achieved a high degree of biochemical differentiation with respect to functions involved in lipid metabolism/transport which may be comparable to that present in 19-21 day fetal rat liver. Further analysis of human and rat fetal liver RNAs using cloned cDNAs should permit construction of a developmental time scale correlating hepatic biochemical differentiation to be constructed between these two mammalian species.     

Increased messenger RNA concentration for carbamoyl-phosphate synthase II in hepatoma 3924A

Previous investigations demonstrated that carbamoyl-phosphate synthase II (synthase II) (EC 6.3.5.5) activity, amount, and in vivo synthetic rate increased approximately 9-fold in the rapidly proliferating rat hepatoma 3924A compared to normal liver. This study provides evidence by Northern and RNA dot blot hybridizations of a 13-fold increase in the amount of hepatoma 3924A synthase II mRNA compared to levels in normal liver. Southern and DNA dot blots indicated amplification of CAD hepatoma 3924A synthase II gene product.