Neuromuscular disuse and atrophy: {\it In vivo\/} magnetic resonance studies of intramuscular lipid metabolism and fluid shift changes

We postulated that neuromuscular disuse results in deleteriously affected tissue-vascular fluid exchange processes and subsequently damages the important oxidative bioenergetic process of intramuscular lipid metabolism. The in-depth research reported in the literature is somewhat limited by the ex vivo nature and sporadic time-course characterization of disuse atrophy and recovery. Thus, an in vivo controlled, localized animal model of disuse atrophy was developed in one of the hindlimbs of laboratory rabbits (employing surgically implanted tetrodotoxin (TTX)-filled mini-osmotic pump-sciatic nerve superfusion system) and tested repeatedly with magnetic resonance (MR) throughout the 2-week period of temporarily induced disuse and during the recovery period (following explantation of the TTX-filled pump) for a period of 3 weeks. Controls consisted of saline/"sham"-implanted rabbit hindlimbs. The validity of this model was established with repeated electrophysiologic nerve conduction testing using a clinically appropriate protocol and percutaneously inserted small needle stimulating and recording electrodes. Evoked responses recorded from proximal (P) and distal (D) sites to the sciatic nerve cuff in the TTX-implanted group revealed significantly decreased (p $<$ 0.001) proximal-to-distal (P/D) amplitude ratios (as much as 50-70% below Baseline/pre-implanted and sham-implanted group values) and significantly increased (p $<$ 0.01) differential latency (PL-DL) values (as much as 1.5 times the pre- and sham-implanted groups). By Day 21 of recovery, observed P/D and PL-DL levels matched Baseline/sham-implemented levels. MRI-determined cross-sectional area (CSA) values of Baseline/pre-implanted, sham- or TTX-implanted, and recovering/explanted and the corresponding contralateral hindlimb tibialis anterior (TA) muscles normalized to tibial bone (TB) CSA (in TA/TB ratios) revealed that there was a significant decline (indicative of atrophic response) from pre- and sham-implanted controls by as much as 20% (p $<$ 0.01) at Day 7 and 50-55% (p $<$ 0.001) at Day 13 of TTX-implantation. In the non-implanted contralaterals, a significant increase (indicative of hypertrophic response) by as much as 10% (p $<$ 0.025) at Day 7 and 27% (p $<$ 0.001) at Day 13 + TTX was found. The induced atrophic/hypertrophic TA muscles were observed to be fully recovered by Day 21 post-explantation as evidenced by image TA/TB ratios. End-point biopsy results from a small group of rabbits revealed comprehensive atrophy of both Type I and Type II fibers, although the heterogeneity of the response supports the use of image-guided, volume-localized proton magnetic resonance spectroscopy (MRS) to noninvasively assess tissue-level metabolic changes. MRS-determined results of a 0.25cc volume of tissue within implanted limb TA muscles under resting/pre-ischemic, ischemic-stressed, and post-ischemic conditions at timepoints during and following disuse atrophy/recovery revealed significantly increased intramuscular spectral lipid levels, as much as 2-3 times (p $<$ 0.01) the Baseline/pre-implanted values at Day 7 and 6-7 times (p $<$ 0.001) at Day 13 + TTX, which approached normal levels (compared to pre- and sham-implanted groups) by Day 21 of post-explanation recovery. (Abstract shortened by UMI.) ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

ROLE OF GLUTATHIONE IN FORMALDEHYDE METABOLISM AND TOXICITY

Glutathione (GSH) is involved in the detoxication of numerous chemicals exogenously exposed or endogenously generated. Exposure to these agents cause depletion of cellular GSH rendering these cells more susceptible to the toxic action of these same agents. Formaldehyde (CH(,2)O) was found to deplete cellular GSH, presumably by the formation of the GSH-CH(,2)O complex, S-hydroxymethylglutathione, and its rapid extrusion into the extracellular medium.^ The metabolism and toxicity of CH(,2)O were determined to be dependent upon cellular GSH in vitro and in vivo. The rate of CH(,2)O oxidation decreased and the extent of toxicity increased when isolated rat hepatocytes or strain A/J mice were pretreated with the GSH-depleting agent, diethyl maleate (DEM). Additional experiments were designed to further study the role GSH plays in detoxication using isolated rat hepatocytes.^ L-Methionine protected against the extent of lipid peroxidation and leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), caused by CH(,2)O in DEM-pretreated hepatocytes, further supporting the protective role of GSH against cellular toxicity. The antioxidants, ascorbate, butylated hydroxytoluene, and (alpha)-tocopherol, were all protective against the extent of lipid peroxidation and leakage of LDH in isolated rat hepatocytes. Whereas L-methionine may be protective by increasing the cellular concentration of GSH which is used to detoxify free radicals or by facilitating the rate of CH(,2)O oxidation, the antioxidant, ascorbate, was protective without altering the rate of CH(,2)O oxidation or increasing cellular GSH levels. These results suggest that the free radical-mediated toxicity caused by CH(,2)O in DEM-pretreated hepatocytes is due to the further depletion of GSH by CH(,2)O and not to increased CH(,2)O persistence. How this further depletion in GSH by CH(,2)O in DEM-pretreated hepatocytes results in lipid peroxidation and cell death was further investigated.^ The further decrease in GSH caused by CH(,2)O in DEM-pretreated hepatocytes, suspected of stimulating lipid peroxidation and cell death, was found not to be due to depletion of mitochondrial GSH but to depletion of protein sulfhydryl groups. In addition, cellular toxicity appears more closely correlated with depletion of protein sulfhydryl groups than with an increase in cytosolic free Ca('2+). The combination of CH(,2)O and DEM may be a useful tool in identifying these critical sulfhydryl-protein(s) and to further understand the role GSH plays in detoxication. ^

IN VITRO METABOLISM OF 6-THIOGUANINE IN RAT SUBCELLULAR FRACTIONS

The metabolism of the antitumor agent 6-thioguanine (TG, NSC-752) by rat liver was studied in vitro. Livers from adult male Sprague-Dawley rats were homogenized and the "liver homogenate" was subjected to differential centrifugation to obtain the "10,000 x g pellet", the "post-mitochondrial fraction", the "cytosol fraction", and the "microsomes". The homogenity of each fraction was estimated by appropriate marker enzyme assays. To delineate the in vitro metabolism of TG by rat liver, 0.2 mM of {8-('14)C}TG was incubated with different subcellular fractions in KCl-Tris-MgCl(,2) buffer, pH 7.4 at 37(DEGREES). The metabolites formed were identified by chromatography, UV spectrometry, as well as mass spectrometry. After a 1 hr incubation, TG was metabolized by the liver homogenate, the 10,000 x g pellet and the post-mitochondrial fraction mainly to 6-thioguanosine (TGR), accompanied by varying lesser amounts of 6-thiouric acid (TUA), allantoin, guanine-6-sulfinic acid (G-SO(,2)H) and an unknown product. In comparison, the cytosal fraction converted TG almost entirely to TGR and TUA in equal amounts. The formation of TGR from TG was limited by the endogenous supply of ribose-1-phosphate. With the microsomal fraction, however, TG was metabolized significantly to G-SO(,2)H and the unknown, accompanied with some TGR. After a 5 hr incubation the metabolism of TG was changed to favor the catabolic route, yielding mostly TUA in the post-mitochondrial and cytosol fractions; but mainly allantoin in the liver homogenate fraction. The kinetic studies of TG metabolism by the subcellar fractions indicated that the formation of TGR served as a depot form of TG. The level of TGR decreased when the catabolism of TG became prominent. The oxidation of TG to GSO(,2)H mediated by the hepatic microsomes represented a new catabolic pathway of TG. This GSO(,2)H, under acidic conditions, readily decomposes to guanine and inorganic sulfate. In the presence of reduced glutathione in Tris buffer, pH 7.8 at 25(DEGREES), GSO(,2)H is adducted to glutathione chemically to form S-(2-amino-purin-6-yl) glutathione and conceivably, inorganic sulfate. Therefore, the formation of GSO(,2)H from TG might have implication in the desulfuration mechanism of TG. On the other hand, the unknown formed from TG by the action of the microsomal enzymes appeared to be a TG conjugate. However, it is neither a glutathione, a glucuronide, nor a ribose conjugate. Additionally, the deamination of TG by guanine deaminase (E.C.3.5.4.3) isolated from rat liver was also investigated. TG is a poorer substrate (Km = 4.8 x 10('-3)M) for guanine deaminase than that of guanine (Km = 4.7 x 10('-6)M) at pH 7.25, optimal pH for TG as a substrate. TG is also a competitive inhibitor of guanine for guanine deaminase, with a ki of 2.2 x 10('-4)M. ^

STUDIES ON ENERGY METABOLISM IN THE HIPPOCAMPUS AFTER ISCHEMIA

The gerbil model of ischemia was used to determine the effect of carotid occlusion on energy metabolites in cellular layers of discrete regions of the hippocampus and dentate gyrus. Levels of glucose, glycogen, ATP and phosphocreatine (PCr) were unchanged after 1 minute of ischemia. However, 3 minutes of ischemia produced a dramatic decrease in net levels of all metabolites. No additional decrease was observed after 15 minutes of ischemia. Re-establishment of the blood flow for 5 minutes after a 15 minute ischemic episode returned all metabolites to pre-ischemia levels. Concentrations of glucose and glycogen were elevated in sham-operated animals as a function of the pentobarbital anesthetic employed. In other studies, elevated GABA levels (produced by inhibiting GABA-transaminase with (gamma)-vinyl-GABA (GVG)) were found to decrease the rate of utilization of the high-energy phosphate metabolites ATP and PCr in the mouse cortex. In addition, glucose and glycogen levels were increased. Thus, tonic inhibition by GABA produced decreased cellular activity. Additional experiments demonstrated the attenuation of ischemia-induced metabolite depletion in cellular layers of regions of the hippocampus, dentate gyrus and cortex after GVG administration. Under ether, 1 minute of bilateral carotid occlusion produced a dramatic decrease in metabolite levels. After GVG treatment, the decrease was blocked completely for glucose, glycogen and ATP, and partially for PCr. Therefore, GABA-transaminase inhibition produced increased levels of GABA which subsequently decreased cellular activity. The protection against ischemia may have been due to (a)decreased metabolic rate; the available energy stores were utilized at a slower rate, and (b)increased levels of energy substrates; additional supplies available to maintain viability. These data suggest that the functional state of neural tissue can determine the response to metabolic stress. ^

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

XYLITOL METABOLISM IN RAT LIVER AND HEPATOCELLULAR CARCINOMAS

The significance of nutritional factors in cancer research has been strongly emphasized. Such research is concerned not only with epidemiological effects relative to dietary factors on the causation of cancer, but with nutritional effects as an energy source on the prevention of cancer. Many studies speculate that the energy flow between tumor and host can be regulated by dietary intake. However, little knowledge on the comparison of the specific nutritional and energy requirements of different cells and tissues is available. Most popular and essential energy sources for the body are the carbohydrates. Among them, xylitol is known as efficient an energy source as glucose. In carbohydrate metabolism, glycolysis is one of the major energy producing pathways. However, recently the existence of an alternate catabolic pathway in mammals for carbohydrate besides glycolysis, i.e. bypass through triosephosphates to lactate via methylglyoxal has been suggested. This bypass was implicated to regulate glycolysis and also be responsible for the fluctuation in the levels of a regulator of cell growth. Methylglyoxal itself is known as a cancerostatic agent. The alterations of biochemical parameters in xylitol metabolism in animals indicated that xylitol may be metabolized through a methylglyoxal pathway.^ To elucidate the biological effect of xylitol as an energy source and the biological effect of its metabolites as a cancerostatis agent, the mode and extent of metabolism must be understood in tumor-bearing animals. Differential utilization of xylitol and glucose, if any, between tumor and host in such animals may exert tissue selective effects on both in terms of methylglyoxal formation and energy provision. The aim of this work was to assess the extent to which the differential utilization of xylitol might be used to generate different metabolic pathways in tumor and host, and to consider a role of nutrition in cancer.^ The results disclose that the existence of a pathway for biological methylglyoxal formation in normal rat liver has been confirmed in single cell suspension; the metabolic significance of the methylglyoxal pathway in the metabolism of glucose and xylitol has been evaluated quantitatively in normal rat liver and the differential metabolism of glucose and xylitol through overall catabolic pathways of carbohydrates has been studied in normal hepatic cells, AS-30D hepatoma and other several hepatoma lines. ^

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

Arabinosylguanine: Metabolism, action, and lineage-specific cytotoxicity in human leukemia cells

9-β-D-arabinosylguanine (ara-G), an analogue of deoxyguanosine, has demonstrated T-lymphoblast selective anti-leukemia activity both in vitro and in vivo in cell lines and primary cells and in phase I investigations. The present work was initiated to identify factors that result in this selectivity. ^ The cytotoxicity of ara-G is manifest only after its phosphorylation. Experiments using cell lines transfected to overexpress specific nucleoside kinases demonstrated that the phosphorylation of ara-G to its monophosphate is by both cytoplasmic deoxycytidine kinase and mitochondria) deoxyguanosine kinase. Ara-G monophosphate is converted to its 5′-triphosphate (ara-GTP) in cells by these kinases and then incorporated into DNA. Mechanistic studies demonstrated that incorporation of ara-GTP into DNA was a necessary event for the induction of cell death. ^ Pharmacokinetic and pharmacodynamic studies utilizing three human acute leukemia cell lines, CEM (T-lymphoblastic), Raji (B-lymphoblastic), and ML-1 (myeloid) were performed. CEM cells were most sensitive to ara-G-induced inhibition of colony formation, accumulated ara-GTP at a faster rate and to a greater degree than either Raji or ML-1, but incorporated the lowest number of ara-G molecules into DNA. The position of incorporation was internal and similar in all cell lines. The terminal elimination phase of ara-GTP was >24 h and similar in these cells. Comparisons between inhibition of colony formation and ara-GTP incorporation into DNA demonstrated that while within a cell line there was correlation among these parameters, between cell lines there was no relationship between number of incorporated ara-G molecules and ara-G(TP)-mediated toxicity suggesting that there were additional factors. ^ The expression of membrane bound Fas and Fast was unchanged in all cell lines. In contrast, there was a 2-fold increase in soluble Fast, which was found exclusively in CEM cells. Ara-G-mediated apoptosis in CEM occurred from all phases of the cell cycle and was abrogated partially by Fas antagonist antibodies. These data suggest that Fas-mediated cell death due to the liberation of sFasL may be responsible for the hypersensitivity to ara-G manifested by immature T-cells such as CEM. The role of Fas in ara-G induced death of acute T-lymphoblastic leukemia cells during therapy needs to be tested. ^

Pharmacogenetics of the human glutathione S-transferase P1 gene in the metabolism and therapeutic efficacy of cis-diamminedichloroplatinum

The human GSTP1 gene has been shown, conclusively, to be polymorphic. The three main GSTP1 alleles, GSTP1*A, GSTP1*B, and GSTP1*C, encode proteins which differ in the 3-dimensional structure of their active sites and in their function in phase II metabolism of carcinogens, mutagens, and anticancer agents. Although, it is well established that GSTP1 is over expressed in many human tumors and that the levels of GSTP1 expression correlate directly with tumor resistance to chemotherapy and inversely with patient survival, the significance of the polymorphic GSTP1 gene locus on tumor response to chemotherapy remains unclear. The goal of this project was to define the role and significance of the polymorphic GSTP1 gene locus in GSTP1-based tumor drug resistance and as a determinant of patient response to chemotherapy. The hypothesis to be tested was that the polymorphic GSTP1 gene locus will confer to tumors a differential ability to metabolize cisplatin resulting in a GSTP1 genotype-based sensitivity to cisplatin. The study examined: (a) whether the different GSTP 1 alleles confer different levels of cellular protection against cisplatin-induced cytotoxicity, (b) whether the allelic GSTP1 proteins metabolize cisplatin with different efficiencies, and (c) whether the GSTP1 genotype is a determinant of tumor response to cisplatin therapy. The results demonstrate that the GSTP1 alleles differentially protect tumors against cisplatin-induced apoptosis and clonogenic cell kill in the rank order: GSTP1*C > GSTP1*B > GSTP1*A. The same rank order was observed for the kinetics of GSTP1-catalyzed cisplatin metabolism, both in cell-free and cellular systems, to the rate-limiting monoglutathionyl-platinum metabolite, which was characterized, for the first time, by mass spectral analysis. Finally, this study demonstrates that both GSTP1 genotype and the level of GSTP1 expression significantly contribute to tumor sensitivity to cisplatin treatment. Overall, the results of this project show that the polymorphic GSTP1 gene locus plays a significant role in tumor sensitivity to cisplatin treatment. Furthermore, these studies have contributed to the overall understanding of the significance of the polymorphic GSTP1 gene locus in tumor resistance to cancer chemotherapy and have provided the basis for further investigations into how this can be utilized to optimize and individualize cancer chemotherapy for cancer patients. ^

Alterations in redox and energy metabolism in Ras-transformed cells: Mechanisms and therapeutic implications

Increasing attention has been given to the connection between metabolism and cancer. Under aerobic conditions, normal cells predominantly use oxidative phosphorylation for ATP generation. In contrast, increase of glycolytic activity has been observed in various tumor cells, which is known as Warburg effect. Cancer cells, compared to normal cells, produce high levels of Reactive Oxygen Species (ROS) and hence are constantly under oxidative stress. Increase of oxidative stress and glycolytic activity in cancer cells represent major biochemical alterations associated with malignant transformation. Despite prevalent upregulation of ROS production and glycolytic activity observed in various cancer cells, underlying mechanisms still remain to be defined. Oncogenic signals including Ras has been linked to regulation of energy metabolism and ROS production. Current study was initiated to investigate the mechanism by which Ras oncogenic signal regulates cellular metabolism and redox status. A doxycycline inducible gene expression system with oncogenic K-ras transfection was constructed to assess the role played by Ras activation in any given studied parameters. Data obtained here reveals that K-ras activation directly caused mitochondrial dysfunction and ROS generation, which appeared to be mechanistically associated with translocation of K-ras to mitochondria and the opening of the mitochondrial permeability transition pore. K-ras induced mitochondrial dysfunction led to upregulation of glycolysis and constitutive activation of ROS-generating NAD(P)H Oxidase (NOX). Increased oxidative stress, upregulation of glycolytic activity, and constitutive activated NOX were also observed in the pancreatic K-ras transformed cancer cells compared to their normal counterparts. Compared to non-transformed cells, the pancreatic K-ras transformed cancer cells with activated NOX exhibited higher sensitivity to capsaicin, a natural compound that appeared to target NOX and cause preferential accumulation of oxidative stress in K-ras transformed cells. Taken together, these findings shed new light on the role played by Ras in the road to cancer in the context of oxidative stress and metabolic alteration. The mechanistic relationship between K-ras oncogenic signals and metabolic alteration in cancer will help to identify potential molecular targets such as NAD(P)H Oxidase and glycolytic pathway for therapeutic intervention of cancer development. ^