tyrosine hydroxylase in neuroblastoma

BIOCHEMICAL MEDICINE: 5. 2%-% ( 1971 :

Tyrosine Hydroxylase in Neuroblastoma’

EUGENE M. JOHNSON, JR.,:’ VICTOR I-I. _\IOKGEXlZC)TH, 111. AND NICOLAS ZENKER

Received October 19, 1 !J7(1

In many patients with neuroblastoma, large amounts of catecholamines and of their metabolites arc escrcted into the urines. .&a!. of these compounds in the urine has been found useful for diagnosis and for follow-up study of patients with this tumor. The elcvatccl urinary- excretion of catecholamines and of their metabolites h\- patients with neuroblastoma is related to its neural crest origin siuccx thcl avnlpathctic nerves and the adrenal medulla have, the same ori$n and also s!&~c~size norepinephrine and epinephrine, respcctivcly.

The initial step in the biosynthesis of uorcpi’lc’phri”‘, is tllc hydroxyla- tion of L-tyrosine to Dopa, which is ceatalyzed Iry tyrosille hydroxylase ( 1). This enzyme has been reported to be the rate-limiting step in norepinephrine synthesis (2) and there is also cvidcnce that this cn- zyme is regulated by feed-back inhibition of norc~pinephrint~ in syrn- pathetically innervated tissues ( 3, 4 ‘).

1 ‘&is study was supported by Grant ‘i’3901) from the .%mcricw (:atlcer Society. Grants CA08726 from the National Cancer Institute, GM15700 from thr Institute of General Medical Service, NS5246 from the National Institute of Neurological

Diseases and Stroke, AM06480 f rom the National Institute of Arthritis and hIetaholic

Diseases, U. S. Public Health Service, Bethesda, 3laryland. ’ Present address: Department of Pediatrics, Kyoto Prefectural lYuiversity of

Medicine, Kyoto, Japan. ’ Present address: Department of Pharmawlo~~. Washington rnircrsity I\ledical

School. St. Louis, Missouri. 4 Abbreviations, in consecutive order: Dopa : 3.3-dihydroxyphenylalaninc. do-

pamine = 3,4-dihydroxyphenylethylamine, HVA =: 3-methoxy-4-hydroxyphrnyl acetic acid, VMA = 3-methoxy-4-hydroxymandelic acid, DhfPH, = 2-amino-4-hydroxy-6,7-

dimethyl-5.6,7,8-tetrahydropteridine, NSD- 1055 .~- m-hydror~-~,-),~~,~ll,~h~n7~.lor;~~-

amine. ., 1

TYROSINE HYDROXYLASE IN NEUROBLASTOMA 23

The elevated excretion of catecholamines and metabolites in neuroblastoma indicates a correspondingly high tyrosine hydroxylase activity, and raises the question of whether there is an abnormality in the tyrosine hydroxylase enzyme itself or in the mechanisms which nor:mally control the activity of this enzyme. Recently tyrosine hydroxylase activity from pheochromocytoma has been reported to be less sensitive to inhibition by catechol compounds than the same enzyme from either normal human adrenal gland, bovine adrenal medulla, or bovine splenic nerve (5, 6).

To date, there have been only a few brief reports on enzymatic tyrosine hydroxylase activity in neuroblastoma and the results of these have been contradictory (7-9).

This paper describes a study of tyrosine hydroxylase activity in two neuroblastoma tumors and its inhibition by catecholamines.

MATERIALS AND METHODS

Tumor I (B.D.) was extirpated at operation from the left adrenal area in a 22-month-old Negro infant who was excreting markedly elevated amounts of Dopa, dopamine,4 HVA,4 and VMA.’ The tumor mass weighed 197 gm, and was very vascular. The tumor tissue was imme- diately placed in a deep-freezer and stored until assayed (11 months). The histological diagnosis of this tumor was neuroblastoma. Heparinized blood specimens were drawn from this patient, plasma was separated by centrifugation and kept frozen until assayed.

Tumor II (C.L.), was obtained at operation from the paravertebral area in a %-year-old Negro girl. This patient excreted catecholamines and metabolites in the normal range. The tumor mass weighed about 3 gm and was highly calcified; on histological examination it was a ganglioneuroblastoma. This tumor was promptly homogenized and assayed for tyrosine hydroxylase activity.

Bovine adrenal tyrosine hydroxylase, prepared by a previously described method ( 1)) was used as a control for inhibition studies of tyrosine hydroxylase by catechols and other compounds.

Tumor tissues were homogenized with 3 vol of phosphate buffer (0.15 M, pH 7) in a glass homogenizer. The homogenate was centrifuged at 105,OOOg for 60 minutes and the supernatant solution was used for the enzyme assay. No attempt was made to remove the endogenous catecholamines or to purify the enzyme preparation. Catecholamines in the tumor tissues were determined as described previously (10). En- dogenous tyrosine content was assayed by the fluorometric method of Wong et al. ( 11). The separation of tyrosine from tyramine in Tumor I (B.D.) was carried out by paper chromatography of the tissue extract.

Chromatograms were developed in the n-butanol-acetic acid-water (4 : I : 1) solvent system.

Tyrosinc hydroxylase activity \\XS assuy~~l 111 triplicate 1,) ;L modifica- tion ( 12) of the procedure described by Nagatsu et nl.. ( 1:; ‘1. The stanci- ard incubation mixture contained :iO !~rnolcs oi r-t\. rosins-S.3-“H (150,000 cpm)?’ 400 !Jmoles acetate buffer (.pN 6.0 i. I.0 ,~.iriolc tctra- hydropteridine ( DMPH,),4 1.0 [mole ferrous iunmoniinn sulfate. 0. I pmole NSD-1055.“9” 90 ,Lmoles mercaptocthanol. 100 or 200 /!.I of solution containing the cnzymt~, and water to ;I final volun~~ of 1 ml. The tubes were incubated for 5-10 minutes at :37 in ;I inetubc)lic shaker. Th(* reaction was stopped by adding 50 ,J of glacial ncctic: ,rcitI, and the rnixture was passed through a 5 x 30-mm column of Do\v(s~ 50 i200-400 mesh. H+ form! which was washed with an additional 1 1111 of distilled water. The effluent and water wash were collectctl in :I counting {ial. 10 ml of Bray’s solution I’ 14) were added. mtl the- tritriatcd uxtc.1 fwnwl hy the reaction was assayed in a liquid-scintillation spcctrorllc~tcr. Quench correction was achieved by the use of automatic cxtcrnal stal~d;lrdizntiorl.

L-Dopa, dopamine, nL-norepinephrinc,. nr,-tr-lnethyltvrosine, ant 1 benzimidazole-5 (6) -nL-alanine dihydrochloride , 12 ) \\‘c’I’(~ I ~cl as inhibitors at concentrations of lo-“--IO-” ~1.

RESI’I,Tb

Most of the enzyme activity of ‘l‘iunor 1 \vas found in the IO5,OOOg supernatant fraction; this was 74.2% of the total enzyme activity. The majority of tyrosine hydroxylase assays in this tumor were done using this supernatant solution.

The endogenous catecholamine and tyrosine contents in the tumor tissue arc presented in Table 1. The tyrosine content of 100 bl,l of supernatant solution was 2.71 nmoles for Tumor I and 4.34 nmoles in the case of Tumor II. Enzyme activities (nmoles of Dopa formed), as be- low, were corrected for these endogenous tyrosine contents.

As shown in Table 2. tyrosine hydroxylase activity in neuroblastoma tissues was not detectable in the absence of DMPH,, i.c.. the tyrosine hydroxylase in neuroblastoma tumor was found to require DMPH, as a cofactor. On the basis of the report by Nagatsu et al. (I ), me used a concentration of lmhs DMPH, in our study; in addition we also con- firmed that tyrosine hydroxylase was inhibited at a higher concentration

’ L-Tyrosine-3,5-3H was purchased frolrl Ambersham/Searle Corpora&In, Arlington

Heights, Illinois. ’ NSD-1055 was kindly supplied by Smith O;r Nephew. Ltd., Harts, F;ngland. ’ Tri-Carb hIode 3375 Liquid Scintillation Spectrometer, Packard Instrument Co..

Downer’s Gove, Illinois.


TABLE 1

CATECHOL COMPOUNDS AND TYROSINE IN NEUROBLASTOMA TUMOR TISSUE

Tumor I (B.D.)

Dopa Dopamine

Norepinephrine Epinephrine

Tumor II (C.L.)

Dopa Dopamine

Norepinephrine Epinephrine

Catechols (fig/pm tissue)

8.2 0.56

0.84 0.029

N.D.,= 0.25

4.6 0.025

Tyrosine (&g/pm tissue)

16.3

25.2

a N.D. = not detected.

( i.e., at 5 mM DMPH, the enzyme activity was 74% of the activity at 1mM).

When DMPH, was added, it was also observed that neuroblastoma tyrosine hydroxylase was activated by Fez+ (1.7-fold in the case of Tumor I and 3.7-fold for Tumor II in the presence of N-3 M Fez+) (Table 2).

In the presence of DMPH, ( W3 M) and Fez+ ( 1O-3 M), Tumor I formed 7.98 nmoles of Dopa/minute/gm tissue, even after 11 months’ storage. This activity is equivalent to 2080 pg of Dopa formed/day/gm tumor. Since this tumor weighed 197 gm, the equivalent in &JO forma-

TABLE 2 EFFECT OF DMPH, AND Fez+ ON NEUROBLASTOMA TYRO~INE HYDROX~LASE ACTIVITY’

Addition Tyrosine hydroxylase activity

(nmoles, Dopa formed)

Tumor I Tumor II (B.D.) (C.L.)

1 None 0 0 2 +DMPH, 0.874 0.183 3 +DMPH, + Fez+ 1.46 0.681

a For the assay, 105,OOOg supernatant solution (equivalent to 25 mg wet weight of tissue) for Tumor I and whole homogenate (equivalent to 28 mg wet weight of tissue) for Tumor II were used. The incubation was for 10 minutes at 37” in a. metabolic shaker. DMPHI and Fez+ were added to give a final concentration of 10-s M.

26 IMASHUKI‘ El- AI.

Ko tyrosine h!;tlroxylasc activit! \\-a~ d~~tt~ctctl iii thcp plasma of that first patient i H.D. ) ~YXW in the presctrrce of DMPlf, and Fc’,. This plasma spwimcn show&l no stimulatory or inhibitor!; cffccts on tyrosinc hydroxylaw activity when added to th(a ~qernutant solution of thr.

tumor from the same patient. A comparison of the inhibition of tyrosin(s hydroxylasc~ I)? catwhol

compounds and other inhibitors is presented in Table 3. T3ovir1c adrenal and neuroblastonra tyrosine hydroxylasc showed A similar tlcgrec of inhibition.

.- -.-~--.- ----- -__. .- -.. - -_-.--

DISCUSSIOiY

The properties of neuroblastoma tyrosine hydroxylasc. rccluirernent of DMPH,, as a cofactor. and stimulation by Fe’!-. x(* quite similar to those of bovine enzyme and pheochromocytoll1,t cwzvInc i 1. C;


In our opinion, the most interesting findings which we have obtained are the results of the inhibition study. In contrast to the reported decreased activity of the pheochromocytoma enzyme to inhibition by catechol compounds (5, S), neuroblastoma tyrosine hydroxylase was found to be inhibited by catecholamines and other inhibitors to the same extent as the bovine adrenal enzyme (Table 3).

When studying the effects of catechols as inhibitors in the presence of Fe”+, the formation of complexes between added Fez+ and catechols possibly occurs ( 15). However, at the concentrations of catechols which we used, no significant difference was observed between the inhibition in the presence and in the absence of Fe*+ (Table 3).

In pheocbromocytoma, large amounts of norepinephrine and epinephrine are stored in the tumor tissue. By contrast, the catecholamine content in neuroblastoma is usually relatively low, as also found in our cases. The low catecholamine levels in neuroblastoma are possibly due to the deficient storage capacity of this tumor ( 16).

The apparent decrease in the sensitivity of tyrosine hydroxylase from pheochromocytoma to end-product inhibition by catecholamines may be at least a partial explanation for the high levels of norepinephrine which accumulates in these tumors. On the other hand, the tyrosine hydroxylase from neuroblastoma, although the results represent only two cases, shows inhibition by catecholamines which is comparable to tyrosine hydroxylase from bovine adrenal gland. Therefore, the sensitivity of tyrosine hydroxylase to feedback inhibition by catecholamines, appears to be inversely related to the accumulation of catechola.mines in these tumors. Since the majority of neuroblastoma patients excrete markedly elevated amounts of VMA and other 0-methylated metabolites in the urine, the high total tyrosine hydroxylase activity in neuroblastoma tumors is probably also due to the removal of inhibition by catecholamine through 0-methylation.

It has been reported that tyrosine hydroxylase activity and norepinephrine synthesis, are controlled in the intact neuron by the intra- neuronal levels of norepinephrine (3, 4). In addition, electrical stimulation of the stellate ganglion has been found to increase norepinephrine synthesis from L-tyrosine-14C, but not from L-Dopa-“H in the rat heart, ( 17)) and elevation of the tissue levels by administration of monoamine oxidase inhibitor markedly decreased norepinephrine synthesis from L-tyrosine-14C, while 3H-norepinephrine formation from L-Dopa-3H was actually increased (18). Th ese findings are consistent with the hypothe- sis that the norepinephrine level in the sympathetic nerves exerts a regulatory effect on synthesis by feedback inhibition of tyrosine hydroxylase.

The level of stored catecholamines is apparently not the only factor in

28 IMASHUKTr IiT AL.

the regulation of synthesis, since the epinephrine levels ill the adrenal medulla remain relatively high, but synthesis proceeds quit-e rapidly both when the splanchnic nerves ar(’ stimlhted ! 19, 21 ! ;~nd when animals are exposed to cold ( 12 1.

The findings reported in this paper suggcast that tyrosiucl il!,drox:)‘lasc in neuroblastoma has a “normal” selksitivit!- to feedback inhibition b; norepinephrine; further investigation n+ll 1~ necessary to c&~blish the mechanism for the increase in the total t!,rosinc hydrox>&c~ activit?,, which appears to be present in the rnajoritv of these tunlorc.

Tyrosine hydroxylase activity was assayed in two neuroblastoma tissues. It was found that the neuroblastoma tyrosine hydroxylase re- quired DMPH, as a cofactor and was stimulated by Fe”“. These properties are quite similar to those reported for bovine and pheochromocytoma enzymes.

Catechol compounds and other inhibitors, at concentrations of 2.5 >: lo-“-5 X 10m4 M, inhibited the neuroblastoma enzyme to the same extent as bovine adrenal tyrosine hydroxylase. This finding suggests that neuroblastoma tyrosine hydroxylase is normally susceptible to feedback inhibition. in contrast to the pheochromocytoma enzymc~ which has been reported to be less sensitive to inhibition by catechols,

Both neuroblastoma and pheochronloc)ltotlla are associated with increased synthesis of catecholamines; in the cast> of pheochromocytoma there are large stores of norepinephrintr in thct turnor. but catecholamine synthesis still continues at an elevated rate, apparently because of a decreased sensitivity to feedback inhibition of the tyrosine hydroxylase in this tumor. On the other hand, in ncuroblastoma the catccholamines appear to exert a “normal” feedback inhibition WI tyrosine hydroxylase, and, therefore, we must look elsewhere to explain the high rate of cat+ cholamine synthesis, such as lowering of the intraccllnlar catecholamine levels by rapid metabolism. Further investigation still be necessary to establish the alteration of control mechanisms in ncuroblastoma.

ACKNOWLEDGblENTS

We thank Dr. Marvin Cornblath, Chief of the Department of Pediatrics, Dr. Priscilla A. Gilman, Pediatric Hematologist. Dr. Earl P. Galleher and Dr. Joseph Insoft, Department of Urology, University of Maryland Hospital for the opportunity

to obtain tumor tissue from patient B.D.: and Dr. John J. White and Dr. Howard Wexler, Division of Pediatric Surgery, Department of Surgery. Johns Hopkins Hospital for tumor tissue from patient C.1,. In addition, we acknowledge with pleasure the assistance of Mr. Gordon Jarman in the c-omputation of the radioactivity

in the tyrosine hydroxylase assay.

TYROSINE HYDROXYLASE IN NEVROBLASTOMA 29

REFERENCES

1. NAGATSU, T., LEVITT, M., AND UDENFFUEND, S., J. Biol. Chem. 239,291O (1964). 2. LEVITT, M., SPECTOR, S., SJOERDSMA, A., AND UDENFRIEND, S., J. Pharmucol.

Exp. Ther. 148, 1, ( 1965). 3. ALOUSI, A., AND WEINER, N., PTOC. Nat. Acad. Sci. U. S. A. 56, 1491 (1966). 4. SEDVALL, G., AND KOPIN, I. J., Pharmacologist 8, 189 ( 1966) ( abstr.). 5. ROTH, R. H., STJARNE, L., LEVINE, R. J., AND GIARMAN, N. J., J. Lab. CZin. Med.

72, 397 ( 1968). 6. NAGATSU, T., YAMAMOTO, T., AND NAGATSU, I., Biochem. Biophys. Acta 198,

210 (1970). 7. GOLDSTEIN, M., in “Recent Results in Cancer Research,” Vol. 2, p. 66. Springer-

Verlag, Berlin, 1966. 8. GOLDSTEIN, M., PROCHOROFF, N., AND SIRLW, S., Experientia 21, 592 (1965). 9. SANPITAK, N., KATHAN, R. H., AND ROSENTHAL, I. M., J. Pediat. 74, 834 ( 1969).

10. IMASHUKU, S., AND LABROSSE, E. H., J. CZin. Endocrinol. Metab. (in press). 11. WONG, P. W. K., O’FLYNN, M. E., AND INOUYE, T., Clin. Chem. 10, 1098 (1964). 12. MILKOWSKI, J. D., MILLER, F. M., JOHNSON, E. M., AND ZENI(.ER, N., J. Med.

Chem. 13, 741 ( 1970). 13. NAGATSU, T., LEVITT, M., AND UDENFRIEND, S., Anal. Biochem. 9, 122 (1964). 14. BRAY, G. A., Anal. Biochem. 1, 279 (1960). 15. UDENFRIEND, S., ZALTZMAN-NIERENBERG, P., AND NAGATSU, T., Biochem.

Pharmucol. 14, 837 ( 19’65). 16. PAGE, L. B., AND JACOBY, G. A., Medicine 43, 379 (1964). 17. GORDON, R., REID, J. V. O., SJOERDSMA, A., AND UDENFRIEND, S., Mol. Pharmacol.

2, 606 (1966). 18. SPECTOR, S., GORDON, R., SJOERDSMA, A., AND UDENFRIEND, S., Mol. Pharmacol.

3, 549 ( 1967). 19. HOLLAND, W. C., AND SCH~~MANN, H. J., Brit. J. Pharmucol. 11, 449 (1956). 20. BYGDEMAN, S., AND VON EULER, U. S., Acta PhysioZ. Stand. 44, 375 (1956).

21. GORDON, R., SPECTOR, S., SJOERDSMA, A., AND UDENFRIEND, S., J. Phawuzcol. Exp. Ther. 153, 440 ( 1966).

tyrosine hydroxylase in neuroblastoma

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