Hepatoma 3924A Cells Research Articles

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D.

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide).

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.

Biochemical pharmacology of acivicin in rat hepatoma cells

The antiglutamine agent acivicin, l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, inhibited the growth of hepatoma 3924A cells in culture. After 7 days of incubation with the drug, an lc 50 of 1.4 μM was observed by determination of colony forming ability. A combination of cytidine (1 mM), deoxycytidine (10 μM) and guanosine (10 μM) completely protected the hepatoma cells against the cytotoxic action of acivicin, but each nucleoside by itself had no effect. Acivicin (0.1 mM) inhibited the incorporation of uridine and thymidine into macromolecules, but not that of leucine. Acivicin depressed the pools of CTP, GTP, dCTP, dGTP and dTTP to 46, 62, 40, 64 and 53%, respectively, but it increased UTP level to 152% of the values of untreated cancer cells. The activity of a highly purified CTP synthetase (EC 6.3.4.2) from rat liver and hepatoma 3924A was inhibited by acivicin. The inhibition was competitive with respect to l-glutamine, and the K i values with liver and hepatoma enzymes, determined by Dixon and reciprocal plots, were 1.1 and 3.6 μM respectively. The hydroxy analog of acivicin was also a competitive inhibitor, but it was less effective than acivicin, with a K i value of 1.8 mM for the hepatoma enzyme. Our observations on the impact of acivicin on the behavior of pools of ribonucleotides and deoxyribonucleotides and the competitive inhibition of purified CTP synthetase from hepatoma cells suggest that a major mechanism of action for this drug is the inhibition of CTP synthetase and GMP synthetase (EC 6.3.5.2).

Rat Hepatomas: Chemotherapy With Lycurim and Pyrazofurin<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Hepatoma 8999 was sensitive to Lycurim [1,4-di-(methylsulfonyloxy-ethylamino)-1,4-dideoxy-ms-erythritol] with a mean lethal dose (LD50) of 8.1 X 10(-8) M for a 6-hour treatment in vitro. The drug dose lethal to 10% of the rats with Lycurim (10 mg/kg) injected ip 12 times into hepatoma 8999-bearing BUF rats at 10-day intervals provided a mean increase in life-span (ILS) of 156%. The more rapidly growing, less differentiated hepatoma 3924A was tenfold less sensitive to Lycurim in vitro, and three treatments in vivo (10 mg/kg given every 8 days) gave an ILS of only 18% in ACI/N rats. Because hepatoma 8999 had a high adenosine kinase activity, the effect of Pyrazofurin (PF; 3-beta-D-ribofuranosyl-4-hydroxypyrazole-5-carboxamide) was examined in vitro: The LD50 was 8.5 X 10(-8) M in a 6-hour exposure. In hepatoma 3924A, with a fifteenfold lower adenosine kinase, the LD50 was 22-fold higher. Three treatments with PF (4 mg/kg given every 2 days) in hepatoma 8999 caused an 18% ILS and no host toxicity, but in hepatoma 3924A no significant ILS was observed. Lycurim combined with PF (0.05 microM each) in hepatoma 8999 cells in vitro provided synergistic kill, but Lycurim and PF (0.3 and 1 microM, respectively) in hepatoma 3924A cells yielded summation. When 10 rats with hepatoma 8999 were treated 15 times with the optimal dose of Lycurim (7.5 mg/kg every 10 1/2 days), 1-year survivors numbered 7. Alternate doses of Lycurim (7.5 mg/kg) and PF (3 mg/kg) at 5-day intervals for 4 months to 10 rats gave an ILS of 152% with eight 1-year survivors and no host toxicity.

Correlation between growth rate and cytochemistry in Morris hepatomas.

The correlation between the cytochemistry (glycoprotein, glycogen, glucose-6-phosphatase, catalase, alkaline phosphatase) and the growth rate of the fast-growing Morris hepatoma 3924A and the slow-growing Morris hepatoma 9618A was studied by utracytochemical techniques. By the chromic acid-phosphotungstic acid technique, acid glycoprotein is stained in glycocalyx, Golgi saccules and vesicles, and secretory granules of the tumor cells of both hepatomas. However, the hepatoma 3924A cells contain thicker glycocalyx and more numerous glycoprotein-rich granules than hepatoma 9618A cells. Abundant alpha and beta glycogen particles are found in hepatoma 3924A. Moderate glucose-6-phosphatase activity is observed in the cisternae of endoplasmic reticulum and nuclear envelope of hepatoma 9618A, but it is totally absent in hepatoma 3924A. High catalase activity is present in numerous peroxisomes of hepatoma 9618A. Hepatoma 3924A contains only a few catalase-positive microperoxisomes. Weak to moderate alkaline phosphatase is present in the plasma membrane and nuclear envelope of hepatoma 9618A cells, while hepatoma 3924A shows no activity of the enzyme. All the cytochemical parameters except glycoprotein show an inverse relationship with the growth rate of the hepatomas. The higher intracellular glycoprotein content of hepatoma 3924A may be related to differences in cell coat secretion (composition and activity) from the slower-growing hepatoma 9618A

Biochemical commitment to replication in cancer cells

The elucidation of the metabolic imbalance that underlies the biochemical commitment to continued replication of cancer cells is essential in the rational targeting of selective anti-cancer chemotherapy. The metabolic strategy of cancer cells entails an ordered pattern of imbalance in the activities of key enzymes and metabolic pathways and in the concentrations of intermediary metabolites, ribonucleotides and deoxyribonucleotides. A meaningful metabolic program was identified which is displayed in neoplastic cells as a result of a reprogramming of gene expression that is manifested in a transformation- and progression-linked fashion. The transition of the non-proliferating, resting cell culture into the proliferating phase provides a particularly suitable biological system of altered gene expression where it is possible to pinpoint the display of enzymic programs that illuminates the biochemical commitment of cancer cells to replication. In the proliferating hepatoma 3924A cells the activity of the pyrimidine synthetic enzymes, ribonucleotide reductase, thymidine kinase, CTP synthetase, uracil phosphoribosyltransferase, OMP decarboxylase, orotate phosphoribosyltransferase and uridine kinase were increased 23-, 13-, 4.9-, 3.1-, 3.5-, 3.6- and 2.1-fold over those of the resting phase cells. Concurrently, the activity of the rate-limiting catabolic enzyme, dihydrothymine dehydrogenase, was decreased to 42%. In consequence, the ratio of the activities of the synthetic to catabolic enzymes of thymidine metabolism increased 31-fold. In purine metabolism, the activity of the rate-limiting enzyme of de novo purine biosynthesis, glutamine PRPP amidotransferase, was increased 1.5-fold and that of the rate-limiting catabolic enzyme, xanthine oxidase, decreased to 75%. In consequence, the ratio of the synthetic to the catabolic enzyme was elevated 2-fold. The activities of the key enzymes of pentose phosphate synthesis and of carbohydrate catabolism rose 1.4- and 1.3-fold, respectively. The extensive rise in the activities of enzymes of pyrimidine and purine biosynthesis indicates that the behavior of these enzymes is more stringently linked with replication than that of other enzymes. The results are in line with earlier ones in this Laboratory on solid tumors indicating an integrated imbalance of the key enzymes of pyrimidine, purine and carbohydrate metabolism that should confer selective advantages to cancer cells. In studying the behavior of purine enzymes that may reveal a relationship with the commitment of cancer cells to replication, the behavior of inosine phosphorylase and guanine deaminase provided such examples. Inosine phosphorylase activity was decreased in all the hepatomas examined irrespective of growth rates of these neoplasms; thus, the decrease of this enzymic activity was transformation-linked. By contrast, the guanine deaminase activity fluctuated in the various lines of the hepatoma spectrum indicating that the behavior of the activity of this enzyme was coincidental to transformation or progression in liver tumors. The relationship of 4 key enzymes with the progression-linked increase in the deoxynucleoside triphosphate pools in hepatomas was examined. The activities of CTP synthetase, thymidine kinase, IMP dehydrogenase and ribonucleotide reductase markedly increased in parallel with tumor growth rates and with the enlargement of the deoxyribonucleotide pools. The applicability of alterations of enzymic programs to 14 different tumor types was shown. There is a shared enzymic program that applies to rodent, avian and human neoplasms. The pattern was observed in carcinomas, sarcomas, leukemias and in human colon tumor xenografts carried in the nude mouse. The biochemical programs displayed were independent from the carcinogenic agent that caused the neoplasms because there was a shared enzymic program in chemically-induced, transplantable rat and mouse tumors, in viral-induced, transplantable avian neoplasms and in primary liver, kidney, lung and colon tumors in human. These observations indicate that strategic aspects of the biochemical commitment of the cancer cells to replication have now been identified. These novel insights should assist in the targeting of selective chemotherapy against cancer cells.

Biochemical programs and enzyme-pattern-targeted chemotherapy in cancer cells

The gene logic of cancer cells was examined by elucidation of the behavior of key enzymes and metabolic pathways in chemically induced, transplantable carcinomas in rodents and in primary tumors in human. In this analysis emphasis was put on the identification of the shared program of enzymic imbalance that was expressed in the various tumors. These studies also pointed out the operation of tumor-type-specific aspects of the neoplastic program. The implications of these enzymic programs for the design of enzyme-pattern-targeted chemotherapy were outlined. The expression of enzymic programs of transformation in pyrimidine metabolism was analyzed. The activities of enzymes of biosynthesis, CTP synthetase, thymidine kinase, uridine kinase, uridine phosphorylase, uridine phosphoribosyltransferase, orotate phosphoribosyltransferase, orotidine 5′-phosphate decarboxylase, and UDP kinase, were increased i all the tumors, whereas the activity of the catabolic enzyme, dihydrothymidne dehydrogenase, was decreased. The behavior of the activities of uridine phosphorylase, which appears to play a synthetic role in liver, and thymidine phosphorylase, a catabolic enzyme, was compared in hepatomas of different growth rates. The growth rate of these solid tumors varied between 52 and 2 weeks. The specific activity of uridine phoshorylase was significantly increase in the 13 liver tumors examined. The activity was particularly high in rapidly growing hepatoma 3683F, being 6-fold higher than that of normal liver. by contrast, the specific activity of thymidine phosphorylase was decreased in all the hepatomas examined. In applying the approaches of enzyme-pattern-targeted chemotherapy, we investigated the action of pyrazofurin and galactosamine in tissue culture and in tumor-bearing animals. Pyrazofurin, a C-nucleoside, inhibited hepatoma 3924A cells in tissue culture, yielding an I 50 = 0.4 μM. Further studies indicated that pyrazofurin was inhibitory to the tumor cells only in the logarithmic phase and not in the plateau phase. In examining the sensitivity of pyrazofurin in different phases of the hepatoma cell cycle, it was demonstrated that this drug exerted a clearcut, phase-specific toxicity. Pyrazofurin was particularly toxic to early G 1 and early S phase cells, but had little or no effect on tumor cells in other phases of the cell cycle. Since our studies showed that in hepatomas the activities of the salvage enzyme, uridine-cytidine kinase, uridine phosphorylase and uracil phosphoribosyltransferase, were markedly increased, it was of interest to determine the ability of various nucleosides to provide protection against pyrazofurin in tissue culture. Uridine or cytidine in concentrations 10- to 100- fold higher than pyrazofurin were able to protect tumor cells from the killing action of pyrazofurin. Uracil had very little protective action and neither did hypoxanthine, a purine metabolite. Pharmacological studies in tissue culture showed that pyrazofurin and galactosamine behaved synergistically in depressing CTP concentration in tissue culture. A similar depression of CTP concentration was also observed in hepatomas 8999 and 3924A carried in rats. The selectivity of this synergistic drug attack was evidenced by the fact that the CTP content was not depressed in the liver of these tumor-bearing animals. Tissue culture studies showed that pyrazofurin did not affect the pools of ATP, dATP, GTP or dGTP, whereas it decreased UTP, CTP, dTTP and dCTP concentrations to 22, 9, 10 and 51%, respectively, of untreated controls. This selective depression of uridylate, cytidylate, dTTP ad dCTP pools provides further insight into the primary action of pyrazofurin in pyrmidine metabolism.