AS-30D Cells Research Articles

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd 2+-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd 2+-produced cytotoxicity. It was found that Cd 2+ induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd 2+. Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd 2+-induced increase in ROS generation. It is concluded that Cd 2+ toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.

Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: The way to apoptosis

Arachidonic acid and, to a smaller extent, oleic acid at micromolar concentrations decreased the mitochondrial membrane potential within AS-30D rat hepatoma cells cultivated in vitro and increased cell respiration. The uncoupling effect of both fatty acids on cell respiration was partly prevented by cyclosporin A, blocker of the mitochondrial permeability transition pore. Arachidonic acid increased the rate of reactive oxygen species (ROS) production, while oleic acid decreased it. Both fatty acids induced apoptotic cell death of AS-30D cells, accompanied by the release of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and association of proapoptotic Bax protein with mitochondria; arachidonic acid being a more potent inducer than oleic acid. Trolox, a potent antioxidant, prevented ROS increase induced by arachidonic acid and protected the cells against apoptosis produced by this fatty acid. It is concluded that arachidonic and oleic acids induce apoptosis of AS-30D hepatoma cells by the mitochondrial pathway but differ in the mechanism of their action: Arachidonic acid induces apoptosis mainly by stimulating ROS production, whereas oleic acid may contribute to programmed cell death by activation of the mitochondrial permeability transition pore.

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0 ± 0.02, 1.3 ± 0.1 and 2.1 ± 0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.

Toxic effects of copper-based antineoplastic drugs (Casiopeinas ®) on mitochondrial functions

To elucidate some of the subcellular and biochemical mechanisms of toxicity of metal-based antineoplastic drugs, mitochondria and cells were exposed to Casiopeinas ®, a new class of copper-based compounds with high antineoplastic activity. The rates of respiration and swelling, the H + gradient, and the activities of succinate (SDH) and 2-oxoglutarate dehydrogenases (2-OGDH) and ATPase were measured in mitochondria isolated from rat liver, kidney, heart, and hepatoma AS-30D. Also, oligomycin-sensitive respiration and ATP content in hepatoma AS-30D cells were determined. Casiopeinas ® (CS) II-gly and III-i inhibited the rates of state 3 and uncoupled respiration in mitochondria. CS II was 10 times more potent than CS III. The sensitivity to CS II was 4–5-fold higher in mitochondria incubated with 2-OG than with succinate. Thus, at low concentrations (≤10 nmol (mg protein) −1; 10 μM), CS II disturbed mitochondrial functions only when 2-OG was present, due to a specific inhibition of 2-OGDH. At high concentrations (≥15 nmol (mg protein) −1), CS II-induced stimulation of basal respiration, followed by a strong inhibition, which correlated with K +-dependent swelling and cytochrome c release, respectively; K +-channel openers induce a similar mitochondrial response. Mitochondria from liver, kidney and hepatoma showed a similar sensitivity towards CS II, whereas heart mitochondria were more resistant. Oxidative phosphorylation and ATP content were also decreased in tumor cells by CS II. The data suggested that CS affected several different mitochondrial sites, bringing about inhibition of respiration and ATP synthesis, which could compromise energy-dependent processes such as cellular duplication.

Plasma membrane Ca2+-ATPase mRNA expression in murine hepatocarcinoma and regenerating liver cells.

The plasma membrane calcium ATPase (PMCA) is an ubiquitous enzyme that extrudes calcium from the cytoplasm to the extracellular space. Four PMCA genes through alternative splicing produce a large diversity of isoforms of this enzyme. We reported previously that the PMCA contained in AS-30D hepatocarcinoma cells showed significant differences in activity in comparison to normal and regenerating liver. In the present study we investigate if the difference in PMCA activity could be related to differential expression of mRNAs encoding different isoforms of PMCA. Using RT-PCR we found that variants 1b, 1x, and 4b are expressed in all liver samples. The hepatoma AS-30 and liver at 2 days of regeneration express low amounts of isoforms 2w, 4b and 4x, and do not express isoforms 4a, 4d and 4z. Fetal and neonatal liver do not express variants 4a and 4d, but they do express variants 4x and 4z. Immunoblot analysis showed a higher ratio ATPase/total protein in the hepatoma AS-30D in comparison to normal liver. Our results suggest that the Ca2+-ATPase kinetic pattern previously observed by us in the AS-30D cells, could be at least partially explained by changes in the mRNA expression of several of the PMCA isoforms expressed in the liver.

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.

Characterization of a new plasma membrane-associated ecto-5′-phosphodiesterase/nucleotide-pyrophosphatase from rat hepatocarcinoma AS-30D cells

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5' phosphodiesterase/nucleotide-pyrophosphatase (5'-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+ stimulated 5'-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [alpha-32P]ATP or [gamma-32P]ATP, respectively, in the absence of any permeabilizing agent.

Potentiation of antitumor immunity by antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to beta-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8(+) but not CD4(+) T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4(+) and CD8(+) T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT.

Substrate Oxidation and ATP Supply in AS-30D Hepatoma Cells

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min × 107 cells)−1), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O2 concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3–0.42).

Characterization of an antineoplastic glucuronide prodrug

The specificity of tumor therapy may be improved by preferentially activating antineoplastic prodrugs at tumor cells pretargeted with antibody–enzyme conjugates. In this study, the conditions required for the efficient activation of p-hydroxyaniline mustard glucuronide (BHAMG) to p-hydroxyaniline mustard (pHAM) were investigated. pHAM induced cross-links in linearized double-stranded DNA at about 180-fold lower concentrations than BHAMG, indicating that the nucleophilicity of pHAM was decreased by the presence of a glucuronide group. The partition coefficient of BHAMG was about 1890 times lower than pHAM in an octanol–water two-phase system, suggesting that the reduced toxicity of BHAMG was due to both hindered diffusion across the lipid bilayer of cells and decreased reaction with nuclear DNA. BHAMG was significantly less toxic to BHK cells that expressed cytosolic Escherichia coli-derived β-glucuronidase (βG) compared with cells that were engineered to secrete βG, demonstrating that extracellular localization of βG was required for optimal activation of BHAMG. The extended retention of mAb RH1 on the surface of AS-30D cells was also consistent with extracellular activation of BHAMG. Taken together, our results indicate that the low toxicity of BHAMG was due to hindered cellular uptake and low alkylating activity. BHAMG must be enzymatically activated outside of tumor cells for maximum cytotoxicity, and non-internalizing antibodies are preferred for human tumor therapy by targeted antibody–enzyme activation of BHAMG.

Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug.

RHI-βG-PEG, formed by linking poly(ethylene glycol)-modified β-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 μg ml−1 RH1-βG-PEG and 20 μM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-βG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-βG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo. © 1999 Cancer Research Campaign

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats.

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.

Cure of malignant ascites and generation of protective immunity by monoclonal antibody–targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting β-glucuronidase (βG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 μg RH1-βG, a conjugate formed between recombinant βG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 μg conjugate per 109 tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 μg/ml, respectively, RH1-βG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 μg RH1-βG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody–βG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-βG and BHAMG. All cured rats were completely protected from rechallenge with 2 × 107 AS-30D cells, indicating that successful treatment of animals induced protective immunity. Int. J. Cancer 73:392–402, 1997. © 1997 Wiley-Liss, Inc.

Quantitative analysis of acetoacetate metabolism in AS-30D hepatoma cells with 13C and 14C isotopic techniques.

Experimental hepatoma cells utilize acetoacetate as an oxidative energy source and as a precursor for lipid synthesis. The significance of ketone body metabolism in tumors lies in the study of tumor-host metabolism and the ketoneMic condition that is often present in cancer patients. The quantitative importance of acetoacetate and glucose was investigated in AS-30D cells with use of 13C and 14C isotopic methods. In addition, the effects of acetoacetate were compared with those of dichloroacetic acid (DCA), an activator of pyruvate dehydrogenase (PDH). The 14CO2 ratio method evaluated the entry of pyruvate into the tricarboxylic acid (TCA) cycle and revealed that acetoacetate diverted pyruvate from PDH to pyruvate carboxylation. In contrast, DCA increased the oxidation of glucose largely through PDH, indicating that PDH is not maximally active in the absence of DCA. Isotopomer spectral analysis of lipid synthesis demonstrated that, in the absence of acetoacetate, glucose supplied 65% of the acetyl-CoA used for de novo lipogenesis. When 5 mM acetoacetate was included in the incubation, glucose was displaced as a lipogenic precursor and acetoacetate supplied 85% of the acetyl-CoA for lipogenesis vs. only 2% for glucose. Thus AS-30D cells have a large capacity for acetoacetate utilization for de novo lipogenesis.

Acetoacetate metabolism in AS-30D hepatoma cells.

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocellular uptake of peptides—I: Carrier-mediated uptake of hydrophilic linear peptides with renin inhibitory activity into isolated rat liver cells

The hepatic uptake of a hydrophilic, cationic linear peptide with renin inhibitory activity [5(4- amino- piperidyl -1- carbonyl) - l- 2,6[ 3H]phenyl- alanyl- β- alanyl- (4 S- amino- 3 S- hydroxy -5- cyclohexyl)- pentan-carbonyl- l-isoleucyl-aminomethyl-4-amino-2-methyl-pyrimidine-citrat] (code number EMD 56133; EMD, E. Merck, Darmstadt) was investigated in isolated rat hepatocytes. EMD 56133 was taken up by isolated rat liver cells in a time-, concentration-, energy- and temperature-dependent manner. The uptake was a combination of diffusion and a carrier-mediated process. EMD 56133 was accumulated 4.5-fold in liver cells. Eighty-three per cent of the accumulated peptide was found in the cytosol, not bound to membrane proteins. Seventeen per cent was associated with membrane proteins after cell fractionation and centrifugation at 100,000 g. The permeability coefficient of the non-saturable uptake of EMD 56133 was P = 1.973 × 10 −6 cm/sec. The kinetic constants for the carrier-mediated transport are K m = 92 μM and V max = 128 pmol/mg × min. Various substrate analogs inhibited the uptake of EMD 56133. AS-30D ascites hepatoma cells and Reuber hepatoma cells did not accumulate EMD 56133. The absence of oxygen or a decreased cellular ATP content blocked the hepatocellular uptake of the renin inhibitor. Temperatures above 20° increased the transport; the activation energy was determined to be A app = 41 kJ/mol. The apparently active uptake of EMD 56133 was not sodium dependent. In contrast, the membrane potential might be a driving force for the transport of the positively charged EMD 56133.

Differential sensitivity of AS-30D rat hepatoma cells and normal hepatocytes to anoxic cell damage.

A substantial fraction of cells present within hard tumors experience extremely hypoxic and hypoglycemic conditions that can lead to phenotypic alterations such as increased metastatic potential and chemotherapeutic drug resistance. Little is known regarding the influence of anoxic aglycemia on tumor cell energy metabolism and viability, and no direct comparisons have been made between the effects of this form of metabolic stress on tumor cells and their tissue of origin. In this study, the effects of in vitro aglycemic incubation under N2 (with or without iodoacetate) on trypan blue exclusion, lactate dehydrogenase release, cell surface blebbing, ATP levels, and mitochondrial respiratory capacity of rat AS-30D ascites hepatoma cells and normal hepatocytes were measured. Under anoxic-aglycemic conditions, the period of incubation during which 50% viability was lost was 2 h for hepatocytes and 6-8 h for AS-30D cells. In contrast, the rate of anoxia-induced loss of ATP was comparable for the two cell types, and mitochondrial damage was actually accelerated in the tumor cells. These findings suggest that tumor cells are more resistant to anoxic cell death because of their greater ability to withstand deenergization and subcellular injury.

Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D Ascites hepatocarcinoma plasma membrane

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca2+, Mg2+)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 mumoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 mumoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca2+/mg/10 min, respectively). Interestingly, the ATP in equilibrium with Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca2+, Mg2+)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP in equilibrium with Pi/mg/min, similar to the one observed fo the enzyme from normal liver (30 nmoles ATP in equilibrium with Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.

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.