Hepatoma 3924A Research Articles

178 KINETIC PROPERTIES OF IMP DEHYDROGENASE PURIFIED FROM RAT HEPATOMA 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, a promising target for cancer chemotherapy, was purified about 5,000-fold to homogeneity from rat transplantable hepatoma 3924A. Sephacryl S-400 gel filtration showed that the molecular weight of the native enzyme protein was about 245,000. SDS electrophoresis indicated that the molecular weight of the subunits was about 60,000. Thus, the enzyme has a tetrameric structure. The double reciprocal plotp for substrate dependence yielded Km values for IMP and NAD+ of 23 and 65 uM, respectively. Excess NAD+ caused substrate inhibition in the purified enzyme. The enzyme requires potassium ions; an apparent Km value for K+ was 7.8 mM. Product-inhibition studies showed that XMP was competitive with IMP (Ki = 136 uM) and noncompetitive with NAD+. NADH exerted uncompetitive inhibition with respect to IMP (Ki = 210 uM) and mixed type with NAD+ (Ki(slope) = 290 uM). This inhibitory pattern suggests an ordered Bi-Bi mechanism for the enzyme reaction in which IMP binds to the enzyme first, followed by NAD+. NADH dissociates from the ternary complex after rearrangement, and finally XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme XMP complex. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

94 CHANNELING OF IMP INTO GUANYLATE SYNTHESIS IN THE GROWTH PROGRAM OF HEPATOMA 3924A CELLS

The purpose was to elucidate the relationship of IMP utilization for de novo guanylate and adenylate synthesis during the display of the proliferative program of hepatoma 3924A cells. Purine compounds in the cells were labeled with [14C] formate or L- [3-14C]serine in a serine-free assay medium, hydrolyzed to purine bases, separated and counted. Under these assay conditions labeling of adenine or guanine was proportionate with incubation time and with number of cells. During the transition of hepatoma cells from plateau phase into logarithmic proliferation the concentration of PRPP increased 14-fold, followed by an 8-fold rise in purine de novo synthesis. In plateau phase cells predominantly adenylates were produced. After replating the resting cells there was a sharp increase in the relative labeling of guanine with a concurrent marked decrease in that of adenine in the late lag phase. The subsequent decline of the relative labeling of guanine was accompanied with the restoration in that of adenine with the increase in the cell density. The striking redirection in the distribution of label from IMP utilization to the preferential synthesis of guanylates during the expression of the biochemical proliferative program of cancer cells underlines the potential significance of this pathway as a target of chemotherapy. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

177 TWO DISTINCT TARGET SITES ON IMP DEHYDROGENASE IN CHEMOTHERAPY

The inhibitory mechanisms of ribavirin 5′-monophosphate, (RMP), SM-108 nucleotide (bredinin 5′-monophosphate, BMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites, ribavirin, SM-108 and tiazofurin, were studied in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. RMP and BMP as well as XMP inhibited competitively with IMP and noncompetitively with NAD+. The inhibition by TAD was similar to that of NADU: uncompetitive with IMP and mixed type with NAD+. Ki values for RMP (0.8 uM), BMP (0.11 uM) and TAD (0.13 uM) were markedly lower than those for XMP (136 uM) and NADH (210 uM), and also the Km for IMP (23 uM) and NAD (65 uM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural metabolites, RMP or BMP with IMP-XMP and TAD with NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from inactivation by IMP or XMP but not by NAD or NADH. Ribavirin and tiazofurin together provided synergistic killing of hepatoma cells in clonogenic assays and the metabolic flux of de novo guanylate synthesis was also synergistically inhibited. The results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin against the 2 different ligand sites of IMP dehydrogenase. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

61 RECOVERY OF THE ACTIVITIES OF IMP DEHYDROGENASE AND GMP SYNTHASE AFTER TREATMENT WITH TIAZOFURIN AND ACIVICIN IN HEPATOMA CELLS IN VITRO

Tiazofurin, a C-nucleoside, and acivicin, a glutamine antagonist, are targeting against de novo guanylate synthesis and are under clinical trials as anticancer drugs. To elucidate the mechanisms of the recovery of the activities of the target enzymes after inhibition by these drugs, hepatoma 3924A cells were incubated either with 100 uM tiazofurin or 10 uM acivicin for three hours and the enzyme activities in cells after replacement with fresh medium were measured. IMP dehydrogenase, inhibited by 86% with tiazofurin, recovered to the control level within two hours. The addition of 6.7 uM cycloheximide did not affect this recovery. GMP synthase activity, which acivicin completely inhibited, recovered to 82% within 36 hours and cycloheximide blocked this return. These results suggest that in tissue culture system the recovery of GMP synthase activity after acivicin treatment is due to the newly synthesized enzyme protein. By contrast, under these in vitro conditions, inhibition of IMP dehydrogenase by tiazofurin is released by other mechanisms, e.g., dissociation of the drug from the enzyme or degradation of the drug. The applicability of the observations obtained in this in vitro system to animals and in human chemotherapy is under investigation. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

Synergistic cytotoxic effect of tiazofurin and ribavirin in hepatoma cells

Tiazofurin, an anti-cancer drug, which induces remissions in human leukemia, and ribavirin, an anti-viral agent, bind at separate sites (NADH and IMP-XMP sites, respectively) on the target enzyme, IMP dehydrogenase. Now we show that the binding to IMP dehydrogenase of these drugs at two separate sites is translated into synergistic inhibition of de novo guanylate biosynthesis and synergistic toxicity in rat hepatoma 3924A cells. These results may be utilized in the chemotherapy of neoplastic diseases and in the treatment of hepatitis virus infection and hepatocellular carcinoma.

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase.

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.

Enzyme-pattern-targeted chemotherapy with tiazofurin and allopurinol in human leukemia

The hypothesis was tested that the increased IMP dehydrogenase activity in human myelocytic leukemic cells, and along with it guanylate biosynthesis, might be a sensitive target to chemotherapy by tiazofurin. 1. 1.|IMP dehydrogenase activity in normal leukocytes was 3.1 ± 0.5 (means ± S.E.)nmol/hr/mg protein and in leukemic cells it was elevated 15- to 41-fold. The activity of guanine phosphoribosyltransferase in normal leukocytes was 389 ± 27 nmol/hr/mg protein and in the leukemic cells it increased 2.8-to 6.8-fold. 2. 2.|IMP dehydrogenase was purified 4,900-fold to homogeneity from rat hepatoma 3924A with a yield of 30%. The kinetic properties of the hepatoma enzyme were similar to those of the enzyme in human myelocytic leukemic blast cells because of the similarity of the K m's for IMP (23 μ m), NAD (44 and 65 μ m); the K i for TAD was 0.1 μ m in both enzymes. 3. 3.|There was a selectivity of the in vitro response to tiazofurin in human normal and leukemic leukocytes. When labeled tiazofurin was incubated with leukocytes from normal, healthy volunteers and from leukemic patients, the leukemic leukocytes made 20- to 30-fold more TAD and the GTP content decreased as compared to normal leukocytes. This procedure proved to be a suitable predictive test in a clinical setting because patients with positive tests resonded to tiazofurin whereas those with negative ones did not. 4. 4.|The National Cancer Institute approved a chemotherapeutic phase I/II trial which concentrates on treatment of refractory acute myelocytic leukemia. Tiazofurin is infused in a 60-minute period with a pump to insure uniform delivery. A novel aspect of the trial was that it was directed primarily by the biochemical impact of tiazofurin on IMP dehydrogenase activity and GTP concentration and the tiazofurin doses were to be adjusted accordingly. Patients received allopurinol as a routine precaution against possible accumulation of uric acid in the kidney. 5. 5.|In the first eight patients, there was one complete remission, two entered the chronic phase, two entered into partial remission, one did not respond, and two were not evaluable. In the five patients who responded, there was a rapid, profound decrease in IMP dehydrogenase activity of the blast cells and a gradual decline in GTP concentrations. The blast cell count followed the decrease in the GTP concentration. The white blood cell count was largely preserved. 6. 6.|Bone marrow aspirates and peripheral blood samples showed that with tiazofurin treatment there was an induced differentiation of the myelocytes. 7. 7.|Allopurinol treatment (300 mg per kg) increased the plasma hypoxanthine concentration from the normal level of about 2 to 3 μ m to 20 to 30 μ m. In presence of low concentrations of guanine in the plasma (less than 0.1 μ m) the 10-fold elevated hypoxanthine concentration would strongly curtail the activity of the salvage enzyme, GPRT. Thus, the inhibition of blast cell IMP dehydrogenase activity by tiazofurin in presence of a markedly diminished GPRT activity caused by the allopurinol treatment resulted in a combination chemotherapeutic impact, leading to the depletion of the blast cell GTP concentration. 8. 8.|Tiazofurin treatment provided hematological remission in five patients with induced differentiation of the bone marrow. Tiazofurin therapy of refractory AML resulted in no leukopenia; therefore, this treatment avoids the morbidity and mortality encountered in conventional chemotherapy of refractory AML. 9. 9.|This clinical investigation provides evidence for the significance of the increased IMP dehydrogenase activity and the elevated capacity of the guanylate biosynthetic pathway in the expression of proliferation and differentiation of human leukocytes.

Transport systems for amphipathic compounds in normal and neoplastic hepatocytes

Photoaffinity labeling of plasma membrane subfractions from liver and of intact liver tissue with a photolabile bile salt derivative, the sodium salt of (7,7-azo-3α,12α-dihydroxy-5β-cholan-24-oyl)-2-aminoethanesulfonic acid, revealed that the hepatobiliary transport of bile salts is accomplished by transport systems different for sinusoidal uptake and canalicular secretion. Polypeptides with apparent M r values 54,000 and 48,000 interact with bile salts at sinusoidal membrane, whereas a polypeptide with an apparent M r of 100,000 is involved in bile salt secretion through the canalicular membrane. Photoaffinity labeling with photolabile derivatives of uncharged and cationic compounds provided evidence that the sinusoidal membrane polypeptides exhibit a broad binding specificity. Photoaffinity labeling studies and kinetic studies suggest that hepatic uptake of different amphipathic anions, uncharged compounds and even of cations is mediated by the sinusoidal transport systems which are involved in the uptake of bile salts. Relatively little is known about the specificity of the canalicular bile salt transport system. The fluorescent bile salt derivative, the sodium salt of { N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 β-amino-7 α,12 α-dihydroxy-5 β-cholan-24-oyl}-2- aminoethanesulfonic acid, is readily taken up into the hepatocytes of all acinar zones and may be used for the evaluation of the functional state of bile salt transport by fluorescence microscopy. Fluorescent microscopic studies with the fluorescent bile salt derivative showed that ascites hepatoma AS 30D cells do not have the ability to take up bile salts and demonstrated the absence of hepatobiliary bile salt transport in the solid Morris hepatoma 7777. Photoaffinity labeling studies revealed that in both tumor cell models, in hepatoma AS 30D and in Morris hepatoma 7777, the plasma membranes were devoid of the polypeptides having affinities to bile salts and amphipathic cations. A slight labeling of bile salt binding membrane polypeptides in plasma membranes from Morris hepatomas 9618A and TC 5123 opens the possibility to study transport in neoplastic hepatocytes.

Simple separation of tritiated water and [ 3H]deoxyuridine from [5- 3H]deoxyuridine 5′-monophosphate in 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 3H 2O, high sensitivity (with a limit of detection of 10 pmol of 3H 2O production), high reproducibility (< ±4.3%), and capability to measure activity in small amounts of sample (30–45 μg protein).

Superoxide generation by the respiratory chain of tumor mitochondria

O ⨪ 2 generation by the succinate oxidase segment of the respiratory chain of mitochondria and submitochondrial particles from hepatoma 22a and hepatoma Zajdela has been studied with the use of the Tiron method. In the presence of succinate, superoxide generation is induced by antimycin, 2- n-4-hydroxyquinoline N-oxide or funiculosin, and is inhibited by mucidin, myxothiazol or cyanide. The rate of O ⨪ 2 generation in the antimycin-inhibited state is maximal at the [ succinate] [ fumarate] ratio of 1:10 and diminishes at more positive and more negative redox potentials. These characteristics of O ⨪ 2-generation are the same as observed earlier in submitochondrial particles from normal tissues. Accordingly, the mechanism of superoxide production is suggested to be the same in tumor and normal mitochondria, namely, autoxidation of the unstable ubisemiquinone in the ubiquinol-oxidizing centre o of cytochrome bc 1 complex. With respect to the rate of O ⨪ 2 generation, the hepatoma mitochondrial membranes are approximately twice as active as bovine heart submitochondrial particles and exceed those from rat liver by more than one order of magnitude.

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.

The modulation of serine metabolism in hepatoma 3924A during different phases of cellular proliferation in culture.

The activities of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase increased markedly during the transition of hepatoma cells from a resting non-proliferating culture into the proliferating growth phase. Activities declined as cells reached confluency and entered the plateau growth phase. This pattern was paralleled by changes in [14C]serine incorporation into nucleic acids. The experiments support the hypothesis that the biosynthesis of serine is metabolically coupled to its utilization for nucleotide precursor formation in cancer cells.

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of denovo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 ± 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Accurate transcription of mouse metallothionein-I gene in a fractionated nuclear extract from a rat hepatoma.

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

Solid tumor models for the assessment of different treatment modalities: XXVI. Estimates of cell survival from tumor growth delay after alternating radiotherapy and chemotherapy.

The doubling time (Td) for hepatoma 3924A in ACI rats is relatively constant between different treatments when growth resumes after treatment with either chemotherapy or radiotherapy. Advantage was taken of this to estimate the fraction of surviving cells (SF) from in vivo growth delay (GD) data using the expression SF = 1/2GD/Td. Survival curves were constructed for several recently published treatment schedules which employed alternating radiotherapy and chemotherapy, and for both daily and multiple fractions per day (MFD) radiation schedules. A doubling time of 5.2 days was assumed, in the range observed for control and regrowing treated tumors, which yields one surviving cell at the TCD37 (3650 cGy single dose). The single fraction, multi-target, single hit model, SF = 1 - (1 - e-D/D0)n with D0 = 406 cGy and n = 1.63 is a reasonable representation of the data over the dose range 375-2250 cGy. The D0 of 406 cGy should be considered as a relative, not absolute value which is dependent on the radiosensitivity of the tumor cells and the accuracy of the doubling time, but is useful in relating the single dose data to more complex radiation schedules. Using D0 = 406 cGy, n = 1.63, and f = number of fractions, the multi-fraction, multi-target, single hit model SF = (1 - [1 - e-D/D0]n)f closely fits our data for 30 daily fractions given at 100 to 375 cGy/day for total radiation doses of 3000 to 11,250 cGy. The fraction of surviving cells for daily radiation alternated with three courses of cyclophosphamide (CP) was in good agreement with an additive effect of the two modalities at radiation doses of 3000 to 5640 cGy. Multiple fractions per day radiation given as 250 cGy fractions was more effective than predicted by the model both when given alone or alternately with cyclophosphamide.

Salvage pathways as targets of chemotherapy

This paper discussed the significance of the activities of purine and pyrimidine salvage enzymes in cancer cells and the targeting against them of chemotherapy. 1. 1.|The activities of salvage enzymes in the rat liver were orders of magnitude higher than those of the rate-limiting enzymes of de novo biosynthesis. A similar relationship was observed in rat hepatomas of different growth rates and in primary colon carcinoma in human. 2. 2.|The concentrations of nucleosides and nucleobases were measured in plasma, liver and hepatoma 3924A in the rat. The freeze-clamp method was required to determine the concentrations of these precursors in rat liver and hepatoma in a reliable and precise fashion because ischemia markedly altered the concentrations of nucleosides, nucleobases and, as shown earlier, nucleotides in these tissues. The results indicated that the liver markedly concentrated the purine precursors, hypoxanthine, guanine and adenine, but not thymidine, which was one-third that of the plasma. Uridine and deoxycytidine occurred in the same concentration as in plasma, but cytidine was 3-fold higher in liver. In the hepatoma in comparison to the liver the concentrations of the nucleosides and bases were altered and for some of the changes the enzymic differences between liver and hepatoma appeared to be accountable. 3. 3.|Kinetic parameters for purine and pyrimidine synthetic enzymes and for the substrates and co-factors were determined in liver and hepatoma 3924A. When enzymic activities were calculated at the tissue steady-state concentrations of the various ligands, the activities of the salvage enzymes were markedly higher than those of the rate-limiting enzymes. 4. 4.|Hepatoma cells were highly sensitive to the action of the transport inhibitor, dipyridamole, in lag and log phases. However, plateau phase cells lost their sensitivity to dipyridamole. 5. 5.|Amphotericin B rendered plateau phase cells sensitive to the inhibitory action of dipyridamole for the incorporation of thymidine. 6. 6.|Amphotericin B enhanced cytotoxicity of dipyridamole in hepatoma and human colon cancer HT-29 cells. 7. 7.|In these studies we discovered the decreased responsiveness to dipyridamole of plateau phase cells and the ability of amphotericin B to restore the sensitivity. Moreover, dipyridamole and amphotericin B were synergistic in their cytotoxic action in rat hepatoma cells and human colon cancer cells. Because human solid tumors have a high concentration of non-growing cells, the action of amphotericin B in rendering cancer cells sensitive to dipyridamole action in combination with inhibitors of de novo purine and pyrimidine biosynthesis is potentially useful in the chemotherapy of human solid tumors.

An unusual NAD(P)H-dependent O2-.-enerating redox system in hepatoma 22a nuclei.

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.

Synergistic effect of coumadin and cytoxan in reducing lung metastases

Lung metastasis is a frequent cancer complication resulting in significant mortality. This study evaluates the effect of coumadin and cytoxan alone and in combination on lung metastases in rats challenged with Morris hepatoma 3924A. Seventy-seven male American Cancer Institute (ACI) rats weighing 200 g were studied. Thirty-seven rats received coumadin orally for six days, which resulted in a prothrombin time 2 times that of controls (30 sec). All rats received 1 × 10 5 clumped Morris hepatoma cells via tail vein injection. Animals were divided into four groups: Group I (controls, n = 20) received no antitumor treatment; group II rats (n = 20) received 25 mg/kg cytoxan intraperitoneally at the time of tumor challenge; group III animals (n = 19) received coumadin alone; while group IV (n = 18) received both coumadin and cytoxan. Rats were evaluated for number of lung metastases and lung weight at 3 weeks postinjection. Data was subjected to statistical analysis by the Student's t test. The mean number of lung metastases were 580 ± 45 in group I, 350 ± 310 in group II, 330 ± 263 in group III, and 200 ± 161 in group IV ( P < .005 [IV] v [I], P < .05 [IV] v [II], [III]), ( P < .05 [II] v [I]), ( P < .05 [III] v [I]). Mean lung weights were 2.597 g ± 1.65 in group I, 2.049 g ± 0.75 in group II, 1.898 g ± 0.80 in group III, and 1.677 g ± 0.31 in group IV. ( P < .025 [IV] v [I], P < .05 [IV] v [II]). These data indicate coumadin and cytoxan act synergistically in reducing lung metastases. These observations suggest the synergistic effect of these two agents may prove useful in the prevention of lung metastases in the clinical setting.

Increased Nucleoside Concentrations in Tumors as Markers of Tiazofurin Action

Abstract Tiazofurin injection (150 mg/kg, i.p.) into rats bearing hepatoma 3924A increased in the tumor the pools of guanine, uridine, thymine, hypoxanthine, inosine, and thymidine 3.5-, 4.4-, 8.0- 18.4-, 18.7- and 42-fold over the controls. There were only minor changes in the host liver. This is the first report showing a selective action of tiazofurin in cancer cells on the concentrations of nucleosides and bases, indicating that these might be used as markers of the impact of tiazofurin in clinical trials.

Amphotericin B renders stationary phase hepatoma cells sensitive to dipyridamole.

This study provides evidence that the dipyridamole inhibitory effect on nucleoside incorporation changed with culture time. Lag and log phase hepatoma 3924A cells were highly sensitive to dipyridamole with IC50 values for thymidine incorporation of 0.20 and 0.31 microM, respectively. In contrast, stationary phase cells were comparatively insensitive to dipyridamole with an IC50 of 38.9 microM. Amphotericin B (10 microM) restored the sensitivity of stationary phase cells to dipyridamole, lowering the IC50 value for thymidine incorporation to 0.25 microM. Amphotericin B also enhanced the cytotoxicity of dipyridamole to hepatoma 3924A cells. The combination of amphotericin B and dipyridamole may be useful in cancer chemotherapy.