Increased Tyrosine Phenol-Lyase Activity in Mice Following Pyridoxal Phosphate Administration1

[CANCER RESEARCH 38, 3663-3667, November 1978] 0008-5472/78/0038-0000$02.00 Increased Tyrosine Phenol-Lyase Activity in Mice following Pyridoxal Phosphate Administration1

Gary W. Elmer,2 Leonard Minor, Gary G. Meadows,3 Darrell H. Spackman, and Vernon Riley

Department ot Pharmaceutical Sciences, School of Pharmacy, University of Washington, Seattle [G. W. Â£., L. U., G. G. U.), and the Pacific Northwest Research Foundation, and The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 [D. H. S., V. Ft.]

ABSTRACT was determined both with and without adding the usual optimal levels of PLP (10 /ng/ml) to the assay mixture. One A limiting factor in the depletion of plasma tyrosine IU of TPL is the amount of enzyme that will catalyze the following tyrosine phenol-lyase injection into normal mice degradation of tyrosine to produce 1 /Â¿molof pyruvate per was found to be the availability of an essential cofactor, min at 37Â°. pyridoxal phosphate. Because of the extremely short half- Mice. Female C57BL/6 x DBA/2 F, (hereafter called life of this cofactor, adequate elevation of circulating BD2F,) mice, 17to 21 g, were obtained from the Charles River cofactor levels for prolonged periods by injection of a Breeding Laboratory, Wilmington, Mass. All mice were pyridoxal phosphate solution was not practical. Similarly, housed in groups of 5 in metal cages with solid floors long-term diets enriched with pyridoxine and pyridoxal covered with hardwood shavings for bedding. The mice phosphate did not significantly improve the efficiency of were equilibrated for at least 7 days prior to use and were the injected holoenzyme. A repository dosage form was given food and water ad libitum except that access to food devised that consisted of an s.c. implant of pyridoxal was denied during the 5 hr prior to obtaining blood sam phosphate suspended in a spermaceti and peanut oil ples. Since the ubiquitous LDH virus, which is present in mixture. Under these conditions a sustained increase in many transplantable tumors (29), can cause significant holoenzyme activity levels and a significant resulting immunological and other alterations in the host (26, 27), all decrease in plasma tyrosine levels were obtained. mice were given injections of the virus 2 to 3 days prior to experimentation as previously described (18), in order to INTRODUCTION assure experimental uniformity. The LDH virus, by itself, does not significantly affect the growth rate of the pig- Interest in extending the application of highly purified mented B-16 melanoma (28). enzymes that selectively degrade specific amino acids in vivo has been stimulated by the utilization of L-asparaginase Analyses. Blood samplesfor the determination of plasma as a chemotherapeutic agent in humans (1, 5-7,10, 22, 41). enzyme and amino acid levels were obtained by orbital Certain glutaminase preparations may also have potential bleeding (25). As tubes of blood were collected, they were for cancer therapy (31, 32, 39). Two PLP"-dependent en immediately chilled in ice water and centrifuged at 0Â°.The zymes, L-methioninase and TPL, have been reported to ex plasma samples were pooled in cups packed in ice; aliquots hibit antineoplastic activity in vivo (14, 18). Both enzymes were then removed for subsequent enzyme assay. The actively degraded their respective amino acid substrates in remainder of the plasma, to be used for amino analysis, was added to a preweighed tube containing 0.6 M sulfo- vivo, but total depletion of these amino acids in the plasma of treated animals has not been achieved. salicylic acid, to precipitate all proteins immediately, thus Dissociation in vivo of the essential PLP cofactor from the inactivating any enzyme activity and stabilizing the amino holoenzymes following injection is an important theoretical acid content. After further preparation, the plasma samples factor in regulating enzyme activity and thus in establishing were analyzed for tyrosine and all other free amino acids the extent and duration of amino acid depletion and, with a modified Beckman Model 120B amino acid analyzer, by the methods of Spackman (35-38). This preparative subsequently, the antineoplastic activity of such enzymes. This report describes the relative instability of holotyrosine procedure has been found to be effective in stopping phenol-lyase in vivo and the enhanced activity of the in vivo plasma enzyme activity in other studies when plasma en zymes were present such as asparaginase (26-30) and enzyme that results when plasma levels of PLP are in arginase (D. H. Spackman, unpublished studies). creased by injections or repository implants. Tumors. Pigmented B-16 melanomas were implanted and measured as described by Meadows era/. (18). MATERIALS AND METHODS PLP. Free plasma PLP levelswere determined by measur Enzyme. Purified TPL was prepared and assayed as ing the relative activation of apo-TPL (19). described previously (18). Where indicated, TPL activity RESULTS 1 These studies were supported in part by Institutional Cancer Grant IN-26 and Grant PDT-73 from the American Cancer Society. 2 To whom requests for reprints should be addressed. Effect of PLP on TPL Activity. In the absence of the 3 Present address: College of Pharmacy, Washington State University, added PLP cofactor, holo-TPL was rapidly converted to the Pullman, Wash. 99163. ' The abbreviations used are: PLP, pyridoxal 5'-phosphate; TPL, tyrosine apoenzyme when injected into normal mice (Table 1). At 5 hr after holoenzyme administration, the plasma was as phenol-lyase; LDH virus, lÃ¡clate dehydrogenase-elevating virus. sayed for the "effective" TLP activity with no PLP added to Received April 6, 1978: accepted July 31, 1978.

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Table 1 In vivo activity of TPL as a function of PLP administration Groups of 5 mice were given injections i.p. of either TPL (200 ID/kg), 10 mw potassium phosphate buffer (pH 7.4), or PLP (50 mg/kg). PLP was reinjected in Groups 2 and 4 again at 2, 3, and 4 hr after the initial treatment. All mice were bled 5 hr subsequent to the initial injections. Plasma samples from all mice within each group were pooled prior to performing the analyses. Single amino acid analyses were performed on each plasma pool. With regular calibration of the amino acid analyzer with calibration mixtures, the results are accurate to better than Â±2%over the range of 10 to 200 nmol/ml (35-38). Plasma TPL activity was determined 5 hr following holoenzyme administration, both without and with PLP (10 /*g/ml) added to the assay mixture in order to compare the effective TPL activity in the host with its potential activity in the presence of optimal cofactor. Replicate assays were performed. TPLGroup1 Plasma

tyrosine level activity activity treatmentBuffer (nmol/ml)74.3 (mlU/ml)0 (mlU/ml)0 Â±1.5" (100)* 2 PLP only 47.5 Â±1.0 (64) 0 0 3 TPL only 31.2 Â±0.6 (42) 40 Â± 1.6 (7) 570 Â±23(100) 4MouseTPL + PLPPlasma10.1 Â±0.2 (14)Effective280 Â±11. (37)Potential760 Â±30(100) " Mean Â±S.E. '' Numbers in parentheses, percentage. the assay mixture. These values were then compared with or pyridoxine were not significantly reduced as compared the "potential" TPL activity in a replicate plasma sample, to to the normal levels of 70 to 80 nmol of plasma tyrosine per which PLP had been added to the optimal level. These ml found in control groups on normal diets. comparative data showed that the effective activity was only Long-Acting PLP Implants. With the goal of substantially 7% of the potential enzyme activity. In comparing Groups 1 increasing plasma PLP levels, a time release repository and 3 in Table 1, it may be seen that the nonsupplemented implant of PLP was devised. A mixture of spermaceti in enzyme was capable of effecting a reduction in plasma peanut oil (30% v/v) was found to be a convenient and tyrosine to 42% of normal. When large amounts of the PLP effective vehicle. This mixture melted at 40-43Â°and could cofactor (50 mg/kg) were injected together with TPL (Group thus be implanted as a liquid. After cooling in situ, it 4), effective enzyme activity levels were substantially ele hardened to a semisolid gel. Release of the PLP suspended vated, to 37% of potential activity. This increased enzyme in the vehicle appeared to be dependent, in part, on the activity was reflected by a corresponding decrease in cir host metabolizing the spermaceti and peanut oil. Surgical culating tyrosine, to 10.1 nmol/ml. PLP administration examinations at 7 days revealed that very little of the alone also resulted in a significant reduction in plasma implant material remained from the i.m. implantation in the tyrosine. This is possibly due to an activation of endoge right hip. nous apotyrosine aminotransferase which also requires Effect of Elevated PLP. Following i.p. administration of this cofactor (43, 44). TPL to PLP-implanted mice, effective plasma enzyme activ PLP Diets. It was previously shown that exogenously ities were increased to 37% of maximal potential activity administered PLP is rapidly cleared from plasma, with a and were maintained at an increased level starting at half-life for free PLP of about 15 min (19). It therefore approximately 24 hr and persisting for at least 100 hr after seemed impractical to elevate plasma PLP levels substan PLP implantation (see Chart 1). The extent of TPL-mediated tially for sustained periods of time by the repeated paren- plasma tyrosine depletion in the mice with PLP implants teral injection of PLP solutions. Studies in humans have was greater (to 8% of normal) than was achieved in mice shown that a daily p.o. supplementation of 20 to 50 mg of supplemented with PLP via i.p. injection (to 14% of normal) pyridoxine produced a 4- to 6-fold increase in plasma PLP, (Table 1) or by p.o. intake. In this PLP implant study, as in from normal levels of 0.01 to 0.03 Â¿Â¿g/rnl(16,21, 34). Dietary the dietary supplementation studies, plasma tyrosine values supplementation was thus explored as a possible means for (70 to 80 nmol/ml) were not significantly reduced in control increasing plasma enzyme activity levels of TPL. Groups of mice receiving only PLP implants. This is not surprising 10 mice were maintained for 7 weeks on either a pyridoxine- when it is considered that PLP administration by diet or enriched diet (10 mg of pyridoxine hydrochloride per g implant would in all probability not achieve as high a peak Purina laboratory chow) or a pyridoxine- plus PLP-enriched cofactor level as the hourly i.p. administration carried out diet (10 mg of pyridoxine hydrochloride plus 1 mg PLP per as described in Table 1. g chow). The results indicated that exogenous PLP or Antitumor Effects. The question of whether the increase pyridoxine in the food did not significantly improve the in effective enzyme levels and the improved tyrosine deple effective enzyme activity of injected TPL under the condi tion observed in mice receiving TPL plus implants of PLP tions used. Free plasma PLP levels were less than 0.1 /*g/ provided a corresponding increase in antimelanoma activity ml in mice on the diets. Also, in contrast to the effect of was examined during 3.5 days of treatment as illustrated in PLP injection on plasma tyrosine (see Group 2, Table 1), Chart 2. It is evident that such short-term administration of the tyrosine levels in mice receiving diets enriched with PLP TPL suppressed the further growth of established B-16

3664 CANCER RESEARCH VOL. 38

TPL Injection Schedule that a greater tumor growth inhibition was produced in IIII mice receiving both TPL and the PLP implant, as compared KX> r25 with mice receiving only TPL. The difference, however, is not highly significant upon statistical examination (p < 0.1 Â»{ for 24 to 144 hr). However, for the "TPL only" mice (Group C), the injected enzyme solution also contained small 15! amounts of PLP to ensure maximal enzyme activity at the time of injection (see Chart 2 legend). io â€¢¿Â§

DISCUSSION The rationale for the therapeutic application of tyrosine- degrading enzymes against pigmented tumors relates to their special sensitivity to tyrosine restriction (2, 3, 11, 18). The ability of TPL to inhibit B-16 melanoma growth was Chart 1. Elevated TPL activity in plasma following implantation of a PLP established in a previous study (18) although pronounced long-acting pellet. At the time shown, mice were given injections of 0.2 ml of a mixture of spermaceti in peanut oil (30% by volume) containing PLP (150 tumor regression was not observed. The data in the present mg/ml). Injections were made i.m. in the right hip. TPL (200 lU/kg) was report show that a major limitation in obtaining maximal injected into separate groups of 3 mice each at 0, 24, 48, 72, and 96 hr. Five activity for TPL in vivo is the continuing availability of PLP hr subsequent to enzyme injection, the mice were bled, and the pooled plasmas were analyzed for both effective and potential (maximal) TPL to the enzyme in the plasma. Under ordinary circumstances, activities, for tyrosine levels, and for free PLP. The effective TPL activities holo-TPL rapidly dissociated into PLP and inactive apoen- are plotted as a percentage of potential (maximal) activities. zyme when injected into mice. With a half-life for free PLP of about 15 min (19), the enzymic consequences are ob vious. Five hr after enzyme injection, the effective plasma levels of active enzyme were consistently less than 11% of i o the potential values. Injection of large amounts of PLP i.p. increased the effective enzyme activity in vivo and improved the enzymic depletion of plasma tyrosine. TPL has a Kmvalue for PLP of 1.5 /AM.This value is of the same order of magnitude as Km's for other PLP-dependent enzymes (4, 8, 9, 12, 13, 15). Lumeng and Li (17) attributed control of free plasma PLP levels to protein binding and to hydrolysis of free PLP by an intracellular alkaline phosphatase. Observations from our laboratories indicate that alka line phosphatase may indeed be a major factor in the rapid dissociation of holo-TPL (20). Such physiological regulation of free plasma PLP levels indicates the basis for some of the difficulties that were encountered in our attempt to elevate free plasma PLP for prolonged periods by dietary 12 24 36 48 60 72 84 supplementation or by i.p. injection. Hours A sustained increase in circulating free PLP levels, as Chart 2. Tumor growth in treated and untreated mice. Influence of PLP evidenced by an increase in TPL activity, was achieved by implants on antimelanoma activity of TPL. Starting at time zero (10 days after tumor inoculation), treated mice (Groups C and D) received TPL (100 ID/kg) means of s.c. implant of the PLP cofactor suspended in a every 12 hr for 7 consecutive doses; each 0.2-ml dose also contained PLP spermaceti peanut oil mixture. The effective enzyme activity (50 mg/ml) in 10 mM potassium phosphate buffer, pH 7.4. Control mice of TPL in mice receiving such implants was dramatically (Group B) were injected on the same schedule but received only the PLP/ buffer solution without enzyme. The mice in Groups A and D received a increased, and this was reflected by a corresponding de single PLP implant at time zero. All mice were given injections of the LDH crease in plasma tyrosine, to 8% of the normal value (Chart virus 3 days prior to tumor inoculation (see text). There were 10 mice in each 1). The failure to eliminate tyrosine completely from the experimental group and each value represents the mean tumor volume of 10 mice. Brackets, S.E. The statistical significance of the values shown is plasma most probably relates to the presumed sluggish discussed in the text. activity of TPL at these low substrate concentrations (Km 0.28 mM) and the still incomplete saturation of the enzyme melanomas. No overt toxicities were apparent in treated with PLP (Chart 1). animals and changes in weight essentially paralleled tumor The increase in plasma TPL activity, was not adequate to growth. Mean tumor volumes in mice treated with enzyme produce a dramatic improvement in antitumor activity when alone (Group C) remained smaller than those in control TPL was tested against the B-16 melanoma. This may be mice in Group B (p < 0.025) from 36 through 84 hr after explained, in part, by the observation that the action of TPL initiation of treatment. Tumors in mice receiving a PLP on growth of B-16 melanoma is primarily cytostatic (18). implant plus enzyme (Group D) were also smaller (p < This is true even in cell cultures where saturating levels of 0.005) than those in Group A controls (implant only) from PLP for TPL were used (G. W. Elmer, unpublished studies). 24 hr through 144 hr after treatment started. These observations suggest that the maximum antitumor In comparing Groups C and D in Chart 2, it may be seen effect to be expected of TPL upon established B-16 mela-

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noma tumors would be a zero growth rate. This was 12. Katunuma. N., Kominami, E., Kobayashi, K.. Hamaguchi, Y., Sanno, K.. essentially achieved with TPL in PLP-implanted mice. To Chichibu, K., Katsunuma, T., and Shiotani, T. Initiating Mechanisms of Intracellular Enzyme Degradation and New Special Proteases in Various exceed this effect and produce tumor regressions may Organs. In: R. T. Schmike and N. K. Katunuma (eds.), Intracellular require a more effective lowering of plasma tyrosine, to Protein Turnover, pp. 186-204. New York: Academic Press, Inc., 1975. substantially below the 5-nmol/ml level that was achieved 13. Kenny, F. T. Properties of Partially Purified Tyrosine-a-Ketoglutarate Transaminase. J. Biol. Chem. 234: 2707-2712, 1959. in these studies. In this regard it would be relevant to know 14. Kreis, W., and Hession. C. Biological Effects of Enzymic Deprivations of what the tyrosine levels were in the tumor tissue relative to L-Methionine in Cell Culture and an Experimental Tumor. Cancer Res., 33: 1866-1869, 1973. plasma levels during therapeutic enzyme treatment. 15. Litwack, G., and Rosenfield, S. Coenzyme Dissociation, a Possible Roberts et al. (33) have examined the activity of phenyl- Determent of Short Half-life of Inducible Enzymes in Mammalian Liver. alanine ammonia-lyase against B-16 melanoma and other Biochem. Biophys. Res. Commun. 52: 181-188, 1973. 16. Lumeng, L., Brashear, R. E., and Li, T. K. Pyridoxal-5'-phosphate in murine tumors. Administration of this enzyme effectively Plasma: Source, Protein Binding and Cellular Transport. J. Lab. Clin. reduced both plasma tyrosine and plasma phenylalanine Med., 84. 334-343, 1974. 17. Lumeng, L., and Li. T. K. Characterization of the Pyridoxal-5'-Phosphate but did not prolong the survival of melanoma-bearing mice. Hydrolase Activity in Rat Liver. J. Biol. Chem.,250. 8126-8131, 1975. Of particular interest was the finding that the tumor levels 18. Meadows, G. G., DiGiovanni, J., Minor, L., and Eimer, G. W. Some of tyrosine and phenylalanine were only partially reduced. Biological Properties and an In Vivo Evaluation of Tyrosine Phenol-lyase on Growth of B-16 Melanoma. Cancer Res., 36. 167-171, 1976. A direct simultaneous comparison of the influence of TPL 19. Meadows. G. G.. Boze, L., and Elmer. G. W. New Rapid Determination and phenylalanine ammonia-lyase on both tumor growth of Pyridoxal Phosphate Using Tyrosine Phenol-lyase. J. Pharm. Sci., 66: rates and survival times would clearly be of interest. 1503-1505, 1977. 20. Meadows, G. G., and Elmer, G. W. Albumin and Alkaline Phosphatase L-Methioninase, another PLP-dependent enzyme, has as Factors Involved in the Regulation of Tyrosine Phenol-lyase Activity. also been reported to exhibit antineoplastic activity in vivo Res. Commun. Chem. Pathol. Pharmacol., 79. 513-527, 1978. (14). Tryptophanase, cysteine desulfhydrase, serine dehy- 21. Miller, L. T., Johnson, A., Bensen, E. M.. and Woodring, M. J. Effect of Oral Contraceptives and Pyridoxine on the Metabolism of Vitamin B,,and dratase, and arginine decarboxylase, all requiring PLP as a on Plasma Tryptophan and a-Amino Nitrogen. Am. J. Clin. Nutr., 28: cofactor, may be of potential therapeutic interest. This 846-853. 1975. 22. Oettgen. H. F.. Old, L. J.. Boyse, E. A., Campbell, H. A., Phillips, F. S., possibility is strengthened by various studies demonstrating Clarkson. B. D.. Tallal, L., Leeper. L., Schwartz, M. K., and Kim, J. H. antitumor effects following restriction of the amino acid Inhibition of Leukemias in Man by L-Asparaginase. Cancer Res.. 27: substrates for these enzymes (23, 24, 40, 42). The data 2619-2631. 1967. 23. Ohnuma.T., Waligunda, J.,and Holland, F. F. Amino Acid Requirements presented in our report indicate that an important experi In Vitro of Human Leukemic Cells. Cancer Res..37: 1640-1644, 1971. mental factor in investigating the potential antineoplastic 24. Regen, J. D.. Vodopick. H., Takeda, S., Lee. W. H., and Falcoun, F. M. properties of PLP-dependent enzymes is the maintenance Serine Requirements in Leukemic and Normal Blood Cells. Science, 763: 1452-1453. 1969. or establishment in vivo of the enzyme-cofactor complex. 25. Riley, V. Adaptation of Orbital Bleeding Technique to Rapid Serial Blood Use of the described long-acting PLP implant, or other Studies. Proc. Soc. Exptl. Biol. Med., 704: 751-754, 1960. 26. Riley, V. Biological Contaminants and Scientific Misinterpretations. repository dosage forms of PLP, may be necessary to Cancer Res.,34: 1752-1754, 1974. evaluate fairly the activity of PLP-dependent enzymes 27. Riley. V. Erroneous Interpretation of Valid Experimental Observations against various tumor systems. through Interference by the LDH-Virus. J. Nati. Cancer Inst., 52. 1673- 1677,1974. 28. Riley, V.. and Spackman, D. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. Increased Tyrosine Phenol-Lyase Activity in Mice following Pyridoxal Phosphate Administration

Gary W. Elmer, Leonard Minor, Gary G. Meadows, et al.

Cancer Res 1978;38:3663-3667.

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