Thymidine and 3'-Azido-3'-deoxythymidine Metabolism in Human ...

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Jun 19, 1990 - chondrial thymidine kinase; PBMC, peripheral blood mononuclear cells; PBL ..... coli, from which the thymidine phosphorylase has been puri-.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267,No. 16, Issue of June 5, pp. 10968-10975.1992 Printed in U.S.A.

Thymidine and 3’-Azido-3’-deoxythymidineMetabolism in Human Peripheral Blood Lymphocytes and Monocyte-derived Macrophages A STUDY OF BOTH ANABOLICANDCATABOLIC

PATHWAYS* (Received for publication, October 23, 1991)

Elias S. J. ArnerSj, Antonio Valentinll, and Staffan ErikssonS From the $Medical NobelInstitute, Department of Biochemistry I, Karolinska Institute, S-104 01 Stockholm and the lDepartment of Virology, Karolinska Institute, S-10521 Stockholm, Sweden

3’-Azido-3’-deoxythymidine(AZT) is HIV-inhibitory in human macrophages, which is surprising in view of the low AZT phosphorylation reported in macrophage extracts. To elucidate the mechanism of AZT activation, we studied AZT anabolism as well as catabolism in human lymphocytes and macrophages, and compared it to that of thymidine. Thymidine kinase (TK)-specific activity in mitogenstimulated lymphocytes was 15 times higher than in macrophages. However, the TK activity per cell was only 1.3 times higher, because of the large macrophage cell volume. Total cellular TK activity, but not specific activity, matched the level of intracellular AZT anabolism. The substrate specificity of TK in macrophages strongly suggests that mitochondrial TK2 was the enzyme phosphorylating thymidine and AZT in these cells, whereas it was cytosolic TK1 in stimulated lymphocytes. In vivo thymidine catabolism was extensive,forming thymine and dihydrothymine. In macrophages more than 95% of the added thymidine (0.5 PM) was degraded within 60 min. AZT, in contrast, was not catabolized, which explains the high AZT nucleotide accumulation, a process opposed only by AZTMP excretion. The lack of catabolism together with phosphorylation by TK2 clarifies how AZT can inhibit human immunodeficiency virus in macrophages. The fact that TK2 and not TK1 phosphorylates AZT in macrophages should have important implications for combination chemotherapy.

The principal drug in clinical use against HIV’ in AIDS

* This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the Medical Faculty of the Karolinska Institute, the Swedish National Board for Technical Development, and Medivir AB. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. § T o whom correspondence and reprint requests should be addressed. Tel.: 46-8-728-69-83;Fax: 46-8-30-51-93. The abbreviations used are: HIV, human immunodeficiency virus; TK, thymidine kinase; TK1, cytosolic thymidine kinase; TK2, mitochondrial thymidine kinase; PBMC, peripheral blood mononuclear cells; PBL, peripheral blood lymphocytes; PBMC+PHA, PBMC stimulated with the mitogen phytohemagglutinin A; M/M, monocytederived macrophages; BMMC, bone marrow mononuclear cells; HPLC, high pressure liquid chromatography; TLC, thin layer chromatography; dThd, thymidine; Thy, thymine; DHT, dihydrothymine; AZT, 3’-azido-3’-deoxythymidine;d4T, 2’,3’-didehydro-2’,3’-dideoxythymidine; AZTMP, AZTDP, and AZTTP, 5‘-mono-, di-, and triphosphates of AZT, respectively.

treatment is the thymidine analog 3‘-azido-3‘-deoxythymidine (AZT). To display anti-HIV activity, it must first be activated by anabolic phosphorylation via thymidine kinase (for review, see Yarchoan and Broder (1989)). There are two thymidine kinases, the cytosolic TK1 which is expressed strictly during S phase and TK2 which is a mitochondrial enzyme constitutively expressed in all eukaryotic cells (Kit, 1985;Bohman and Eriksson, 1990).In proliferating cells, TK1 is at least 20-fold more active than TK2. HIV infection of M/M plays an essential role in the development of AIDS (Gartner et al., 1986a, 1986b; Koenig et al., 1986; Petit et al., 1988), which is why studies of AZT metabolism in these cells are of importance. AZT metabolism in M/ M is most likely different from that in proliferating cells, since mature M/M are truly resting cells that supposedly do not express TK1 but should contain TK2 activity. Recently, comparisons of purified TK1 and TK2 showed that while TK1 phosphorylates AZT about 50% as efficiently as it does dThd, TK2 phosphorylates AZT only about 5% as efficiently as it does dThd (Munch-Petersen etal., 1991; Eriksson et al., 1991). Perno et al. (1988) haveshown that AZT inhibits HIV replication in M/M, even at lower concentrations than in proliferating H9 cells. The positive anti-HIV effect of AZT in infected M/M was explained by a low level of dTTP in these cells, which would enhance the AZTTP antiviral effect. Furman et al. (1986) have shown previously, in proliferating H9 cells, that AZT is phosphorylated by the cytosolic thymidine kinase TK1 andthatthis phosphorylation remained unchanged when cells wereinfected with HIV. In viewof a probable lack of TK1 concurrent with the surprisingly good anti-HIV effect of AZT in M/M, we wished to determine which thymidine kinase phosphorylates AZT in these cells and if this phosphorylation is altered when the cells are infected with HIV. Furthermore, we studied the intracellular metabolism of AZT in M/M, PBL,and in PBMC+PHA, and compared it with that of dThd. MATERIALS AND METHODS AND RESULTS’

Kinase Actiuities-Table I shows the specific activity of TK in cell extracts. It was low in resting cells and increased 16fold when PBMC were stimulated with PHA. It was 50 times higher in PBMC+PHA than in PBL, and itwas at about the same level in M/M as in PBMC. When the cells were infected with HIV, dThd andAZT phosphorylation increased 100% in Portions of this paper (including “Materials and Methods,” part of “Results,” and Figs. 5-8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of astandard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

10968

Catabolism Anabolism and AZT anddThd TABLEI Thymidine kinase specific actiuity in cell extracts Cells and kinase assays were prepared and performed as described under “Materials and Methods.” Results are the mean f S.D. of two to five experiments with three measurements in each and are the same experiments as those in Fig. 1. M/M+HIV shows the combined values of three M/M cultures infected with H I V A and ~ ~ ~three with HIVle.,, between which there was no difference in kinase activity.

10969

PBMC+PHA where no accumulation of intracellular catabolites was observed (Fig. 2d). In both PBL and PBMC+PHA, dTTP and dTDP remained at a constant level throughout the time of incubation (Fig. 2, b and d). There was, however,a marked difference in the level of dTMP when PBL was compared with PBMC+PHA, with no detectable dTMP in PBLbut dTMP being the major metabolite in PBMC+PHA at 30 min, thereafter decreasing Substrate by time (Fig. 2 4 . 10 pM dThd 20 pM AZT In M/M the catabolism of dThd was even greater than in pmolfmgfmin PBMC+PHA or PBL, with more than 95% of the added dThd 4.8 f 0.8 lo0 PM dThd inBMMC, a difference which the authors did not discuss. This difference is however clarified by the fact that TK2 is the AZT phosphorylating enzyme in M/M, whereas TK1 is found in proliferating leukocytes, such as BMMC. Since TK2 is less efficient than TK1 in AZT phosphorylation, but dThd is a very good inhibitor for both TK1 and TK2 (Munch-Petersen et al., 1991),the inhibition of AZT phosphorylation by dThd will be more pronounced in cells that only contain TK2. The 2-fold increase in TK activity and 5-fold increase in AZT phosphorylation in HIV-infected PBMC as compared with uninfected PBMC suggest an induction of TK1, which is also supported by the reduced inhibition of d4T in PBMC+HIV as compared with uninfected PBMC. The most likely explanation is that theHIV source, which wasa supernatant of HIV-infected cell cultures, contained a mitogen-like stimulatory factor. There was, however, noinduction of TK1

and dThd

AZTAnubolism Catabolism and

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Time o f incubation [min] FIG.2. dThd metabolites in PBL and PBMC+PHA. PBL (panelsa and b ) and PBMC+PHA (panels c and d ) were incubated with 3H-Thd (0.5 p ~ 25, Ci/mmol) as described in the text. The results are from TLC analysis of metabolites in the medium (panels a and c ) and HPLC analysis of intracellular metabolites (panels b and d ) . Values are the means of two separate incubations, with all cells from the same donor. 0-0, dThd catabolites; A-A, dTMP; 0-0,dTDP; 0-0, d T T P 0--Q, dThd; 0- -0,Thy; A- -A,D H T 0- -0,X .

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in HIV-infected M/M; thus, TK2is the sole thymidine kinase in HIV-infected M/M, as it is in uninfected M/M. How nucleotides synthesized by TK2, a mitochondrial enzyme, can be utilized in thenucleus (as in DNA incorporation of 3H-TdR during studies of repair in resting cells) or the cytoplasm (AZT nucleotide inhibition of HIV replication in M/M) is not well understood. Two mechanisms seem most probable; (i) nucleotides formed in the mitochondria can be transferred tothe cytoplasm, perhaps via the ATP/ADP transport system, or (ii) TK2 is present in the cytosol in addition to its mitochondrial location, as has been suggested earlier (Munch-Petersen etal., 1991). dThd Metabolism-We found a high catabolism of dThd, and asreviewed by Niemann and Berech (1981), dThd catabolitescan be further metabolized intoavast number of metabolites, such as amino acids, carbohydrates and lipids. The accumulation of intracellular catabolites from dThd that was found in PBL,as well as theappearance of an unidentified metabolite in the medium, might be signs of a further metabolism of the dThdcatabolites. The label in alldThd nucleotides (dTMP,dTDP,and dTTP) were at constant levels throughout the 720-min incubation with PBL, with dTTP about 5 times higher than dTDP and very low dTMP. This was in contrast to that found in PBMC+PHA, where dTMP was the major anabolite of dThd,

decreasing throughout the incubation time. This indicates that in PBMC+PHA, with high TK1 activity and also high de m u 0 synthesis, the rate-limiting step in dThd salvage is not TK1, but rather the thymidylate kinase. The only major metabolite seen in M/M was DHT, except at the earliesttimepoint where Thy and dThd could be detected in themedium, and dTTP intracellularly. It has been pointed out earlier that an extensive catabolism of dThd might give underestimates when dThd incorporation into DNA is used as ameasure for cell proliferation (Bodycote and Wolff, 1986) or for estimation of unscheduled DNA synthesis (Pero et al., 1984). Based on the results in this study, the latter should be especially true for M/M. AZT Metabolism-The -40-fold higher AZTMP level after 360 min in PBMC+PHA compared with PBL was as expected, since PBMC+PHA express a high level of TK1, and the finding confirms earlier studies in this laboratory (Tornevik et al., 1991). In M/M, the 360-min level of AZTMP per cell was 170% of that found in PBMC+PHA. This is in great contrast t o the TKspecific activity pattern, but itcorrelates with the TK activity per cell. The intracellular dTTP pool also has to be considered; since the intracellular dTTP concentration is much higher in lymphoblasts than in M/M (Terai etal., 1991), there is most likely a higher feedback inhibition of thymidine

10972

dThd andAZT Anabolism and Catabolism

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T i m e o f i n c u b a t i o n[ m i n ] FIG.3. AZT metabolites in PBLandPBMC-PHA. PBL (panel a) and PBMC+PHA (panel b ) were incubated with [3H]AZT (0.5 p ~ 11 , Ci/mmol) as described in the text. The results are from HPLC analysis of intracellular metabolites. Values are the means of two separate incubations, performed simultaneously with the dThd incubations shown in Fig. 2 (aand b ) and with cells from the same donor. Please note that the left scale in each panel shows cpm/103 cells, while the right shows pmol/106 cells. A, AZTMP; 0, AZTDP; 0, AZTTP. kinases in PBMC+PHA than in M/M, a fact which would explain why the difference in actual salvage of thymidine or thymidine analogs between PBMC+PHA and M/M will be lower than thedifference in kinase activity in assays utilizing cell extracts. The total amount of AZT anabolites found after 60 min of incubation with M/M is 4.52 pmol/106 cells, which yields a total AZT phosphorylation of 0.075 pmol/min/106 cells. The ratio of the i n vivo phosphorylation during incubation with M and the i n vitro kinase activity using 20 p~ AZT 0.5 ~ L AZT is thus 0.083, which supports the viewof TK2 in M/M as being sufficient for the AZT anabolism seen. The reason for the much higher levels of AZT anabolites in M/M as compared to PBLis most likely due to thehigher specific activity of TK2 in M/M in combination with the manifold larger cell volume. The intracellular decrease of AZTMP at later time points in M/M with the simultaneous increase of AZTMP in the medium resembles what has been seen earlier in CCRF-CEM cells (Fridland et al., 1990) and 3T3 cells (Karlsson, 1990). This AZTMP excretion counteracts the very high levels that otherwise would be the result of an anabolism unopposed by catabolic reactions. It should be pointed out that theprocess of AZTMP excretion can never be seen in i n vitro studies of enzyme activities. Concluding Remarks-Studies of the anabolism of nucleotides and analogs have usually been concerned with the relation between the salvage pathway and de novo synthesis (Bohman and Eriksson, 1990; Cohen et al., 1983; Reichard, 1988; Terai et al., 1991),while studies of the catabolism have been focused on the degradation of dThd in platelets (Desgranges et al., 1981, 1982; Pero et al., 1984) or Escherichia coli, from which the thymidine phosphorylase has been puri-

fied and recently crystallized (Cook et al., 1987; Walter et al., 1990). In one study of i n vitro enzyme activities, 3”O-methyluridine was found to be less than 1%as good a substrate for the pyrimidine phosphorylase as uridine (Krajewska et al., 1978), which agrees with the lack of AZT catabolism observed by us. Also, in a study using PBMC+PHA and the Raji cell line, it has been shown that human lymphocytes lack the capacity of efficient nucleoside synthesis from pyrimidine bases (Perignon et al., 1987). This explains why in M/M, where the catabolism completely depleted the medium of dThd, Thy or DHT were not reutilized to form dThd. In addition to i n vitro measurements, there havebeen studies on whole animal metabolism of pyrimidines. It has been shown that complete degradation to @-alanine from uracil in the rat only takes place in the liver and, to a lesser extent, in the kidney (Holstege et al., 1986). This agrees well with our finding of DHT as end product in leukocyte catabolism, except perhaps in PBL where we found a catabolite not yet identified that could possiblyrepresent further metabolism of DHT. In mouse, 30 min after intravenous injection of dThd, a total of 10-20% was anabolized to nucleotides in different tissues, while the rest (80-90%) was catabolized (Moyer et al., 1985). Bodycote and Wolff (1986) have shown that there exists an interindividual variation in dThd catabolism, and a study from our group has shown a substantial inter- and intraindividual variation in AZT nucleotide accumulation (Tornevik et al., 1991). This poses a problem if levels of intracellular nucleotides are compared between different donors or cell separations. In our study this problem is avoided, since the comparison between dThd and AZT metabolism was performed in cells from the same donor, separated andincubated at thesame time.

dThd and AZTAnabolism and Catabolism

10973

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Time o f i n c u b a t i o n[ m i n ] FIG. 4. AZT metabolites in M/M. HPLC analysis of intracellular metabolites after incubation with [3H]AZT (0.5PM, 11 Ci/mmol). The incubation was done in the same cells as those where dThd was completely catabolized, as described in the text.A, AZTMP; 0 , AZTDP; 0,AZTTP. Also shown is AZTMP (A)as detected in the medium using TLC.

We could not detect any catabolism of AZT, which is in be used in combination therapy with AZT, it may have syngreat contrast to that seen with dThd. We have not found ergistic or additive effect in replicating lymphocytes containany published report on the catabolism of AZT despite the ing TK1 but will inhibit the AZT phosphorylation in M/M extensive literature on the biochemical pharmacology of di- containing TK2 but not TK1. deoxynucleoside analogs. Studies have been focused on anaREFERENCES bolic reactions or elimination routes via liver or kidneys Balzarini, J., Cooney, D. A., Dalal, M., Kang, G. J., Cupp, J. E., (Balzarini et al., 1987,1988; Yarchoan and Broder, 1989; DeClercq, E., Broder, S., and Johns, D. G. (1987)Mol. Phurmmol. Yarchoan et aZ., 1989), but not on the leukocyte intracellular 32,798-806 catabolism. Balzarini, J., Pauwels, R., Baba, M., Herdewijn, P., de Clercq, E., The triphosphate level of dideoxynucleoside analogs has Broder, S., and Johns,D. G. (1988)Biochem. Pharmacol. 37,897903 been shown to correlate to the anti-HIV effect (Ha0 et al., 1988). It is clear from our study that thelevel of intracellular Bodycote, J., and Wolff, S. (1986)Proc. Natl. Acad. Sci. U. S. A. 83, 4749-53 catabolism is of great importance for the accumulation of Bogenhagen, D., and Clayton, D. A. (1976)J. Biol. Chem. 251,2938antivirally active triphosphates. If an analog would be catab2944 olized at a rate as high as that for dThd, the level of phos- Bohman, C., and Eriksson, S.(1990)Biochem. (Life Sci. Adu.) 9,ll35 phorylated analog metabolites would be low, even if the phosBols, N. C., Bowen, B.W., Khor, K.G., and Boliska, S. A. (1980) phorylation was much higher than that of AZT. The ratio of Anal. Bwchem. 106,230-237 kinase/phosphorylase activity for a certain nucleoside analog Browne, M. J., presenting for the Brown University AIDS Program, is thus most likely of great importance for the anti-viral or Clinical Trial Group (1990)in Proceedings of VZth Znternational chemotherapeutic properties of that analog, since the phosConference on AIDS, Sun Francisco, June 19-24, 1990,p. 200 phorylase reaction, in contrast to the nucleotidase reaction, Clayton, D. A. (1982)Cell 28,693-705 Cohen, A., Barankiewicz, J., Lederman, H. M., and Gelfand, E. W. is virtually irreversible. (1983)J. Biol. Chem. 258,12334-40 The fact that TK2 phosphorylates AZT in M/M should Cohn, Z. A., and Benson, B. (1965)J. Enp. Med. 121,153-170,279have implications for combination chemotherapy. As an ex297 ample, d4T has been shown to display high anti-HIV activity Cook, W. J., Koszalka, G. W., Hall, W. W., Burns, C. L., and Ealick, S. E. (1987)J. Bwl. Chem. 262,3788-9 (Hamamoto et al., 1987) and a phase I trial for AIDS treatment with d4T has started(Browne, 1990). It has been shown Desgranges, C., Razaka, G., Rabaud, M., and Bricaud, H. (1981) Biochim. Biophys.Acta 654, 211-8 that d4T is phosphorylated neither by TK1 nor by TK2 Desgranges, C., Razaka, G., Rabaud, M., Picard, P., Dupuch, F., and (Munch-Petersen et al., 1991) and could conceivably be tried Bricaud, H. (1982)Biochem. Phurmacol. 31, 2755-9 for chemotherapy together with AZT. However, if d4T would Eriksson, S., Kierdaszuk, B., Munch-Petersen, B.,Oberg,B., and

dThd and AZT Anabolism and Catabolism Johansson, N. G. (1991) Biochem. Biophys. Res. Commun. 176, 586-92 Fink, R. M., McGaughey, C., Cline, R. E., and Fink, K.(1956) J. Biol. Chem. 2 1 8 , 1-7 Fridland, A., Connelly, M. C., and Ashmun,R. (1990) Mol. Pharmacol. 37,665-70 Furman, P. A., Fyfe, J. A., St Clair, M. H., Weinhold, K., Rideout, J. L., Freeman, G. A., Lehrman, S. N., Bolognesi, D. P., Broder, S., Mitsuya, H., and Barry, D. W.(1986) Proc. Natl. Acad. Sci. U. S. A. 83,8333-7 Gartner, S., Markovits, P., Markovitz, D. M., Kaplan, M. H., Gallo, R. C., and Popovic, M.(1986a) Science 2 3 3 , 215-9 Gartner, S., Markovits, P., Markovitz, D. M., Betts,R. F., and Popovic, M. (1986b) J. Am. Med. Assoc. 2 5 6 , 2365-71 Hamamoto, Y., Nakashima, H., Matsui, T., Matsuda, A., Ueda, T., and Yamamoto, N. (1987) Antimicrob. Agents Chemother.31,90710 Hao, Z., Cooney, D. A., Hartman, N. R., Perno, C. F., Fridland, A., DeVico, A. L., Sarngadharan, M. G., Broder, S., and Johns, D. G. (1988) Mol. Pharmacol. 3 4 , 431-5 Holstege, A., Pausch, J., and Gerok,W. (1986) Cancer Res. 46,557681 Ives, D.H., and Wang, S. M. (1978) Methods Enzymol. 51,337-45 Karlsson, A. (1990) I n Vitro Studies of the Metabolism of Antiuiral Nucleoside Analogues. Thesis, Karolinska Institutet, Stockholm Kit, S. (1985) Microbiol. Sci. 2, 369-375 Koenig, S., Gendelman, H. E., Orenstein, J. M., Dal Canto, M. C., Pezeshkpour, G. H., Yungbluth, M., Janotta, F., Aksamit, A., Martin, M. A., and Fauci, A. S. (1986) Science 2 3 3 , 1089-93 Krajewska, E., De Clercq, E., and Shugar, D.(1978) Biochem. Phar~ U C O2~7 ., 1421-1426 Mitsuya,H.,Yarchoan, R.,Kageyama, S., andBroder, S. (1991) FASEB J. 5 , 2369-81

Moyer, J. D., Malinowski, N., and Ayers, 0. (1985) J. Biol. Chem. 260,2812-8 Munch-Petersen, B., Cloos, L., Tyrsted, G., and Eriksson, S. (1991) J. Biol. Chem. 266, 9032-8 Niemann, M. A., and Berech, J. (1981) Biochim. Biophys. Acta 652, 347-53 Perignon, J. L.,Bories, D. M., Houllier, A.M., Thuillier, L., and Cartier, P. H. (1987) Biochim. Biophys. Acta 9 2 8 , 130-6 Perno, C. F., Yarchoan, R., Cooney, D. A., Hartman, N. R., Gartner, S., Popovic, M., Hao, Z., Gerrard, T. L., Wilson, Y. A., Johns, D. G., and Broder, S. (1988) J. Exp. Med. 168, 1111-25 Pero, R. W., Johnson, D., and Olsson, A. (1984) Cancer Res. 44, 4955-61 Petit, A. J., Tersmette,M., Terpstra, F. G., de Goede, R. E., vanLier, R. A., and Miedema, F. (1988) J. Zmmunol. 1 4 0 , 1485-9 Pogolotti, A. L., Nolan, P. A., and Santi, D.V. (1981) Anal. Biochem. 117,178-86 Posakony, J. W., England, J. M., and Attardi, G. (1977) J. Cell Biol. 74,468-91 Reichard, P. (1988) Annu. Reu. Biochem. 5 7 , 349-74 Spyrou, G., and Reichard, P. (1987) J . Biol. Chem. 2 6 2 , 16425-32 Sundqvist, V. A., Albert, J., Ohlsson, E., Hinkula, J., Fenyo, E. M., and Wahren, B. (1989) J. Med. Virol. 2 9 , 170-175 Szebeni, J., Patel, S. S., Hung, K., Wahl, L. M., and Weinstein, J. N. (1991) Antimicrob. Agents Chemother. 35, 198-200 Terai, C., and Carson, D. A. (1991) Exp. Cell Res. 1 9 3 , 375-81 Tornevik, Y., Jacobsson, B., Britton, S., andEriksson, S. (1991) AIDS Res. Hum. Retroviruses 7 , 751-759 Walter, M. R., Cook, W. J., Cole, L. B., Short, S. A., Koszalka, G. W., Krenitsky, T . A., and Ealick, S. E. (1990) J. Biol. Chem. 2 6 5 , 14016-22 Yarchoan, R., and Broder, S. (1989) Pharmacol. Ther. 40,329-48 Yarchoan; R., Mitsuya, H., Myers, C. E., and Broder, S . (1989) N. Engl. J. Med. 3 2 1 , 726-38 Zhu. Z.. Schinazi. R. F.. Chu. C. K.. Williams. G. J.. Colbv. C. B.. and Sommadossi, J. P. (1990) Mol. Fhrmacol. '38, 929-38

Supplemental Material to

METABOLISM OF T H Y M I D W E A N D3'-AIIDO-3"DEOXYTHYMIDINE1AZT) IN I1UMAN LYMPHOCYTES A N D M/M: A S T U D Y OF BOTH ANABOLIC AND CATADOLIC PATHWAYS. Eliar S J A m e r . Antonio Palentin and Stattan Eriksbon

dThd andAZT Anabolism and Catabolism Typical and lnturmative eranpler at t h e HPLC rhronatanrana and the &dentifiration 01 m e t a h l i t e s using plritlons of standards a r e s h a m in tin. e . m e first peal. in the chromato s r m ~a t t r r incubation r l t h dThd contained co-eluted nuclsoride and catabolites.

a

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Fie 7. TLC chromatogram of medium from PBL incubated with %-dThd. '[LC was run, cut and counted as described ~n Materials and Methods. The fimre shorr a n a l w i s of 50 #i of the medium attrr dTt8d incubation for 720' with l'BL. Shorn i r ai," the Posltion o t standards (shaded a r e a s ] . m e f i r s t a r r o w indicate. were sample was applied and the second arrow t o where the TLC solution ~1)sallored to ascend.

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F i n 8. l U chr-topram of medium f r a 720' of AZT i n e u h tion with U r n . F o r explanation. see leuend t o fig. 8.

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