[CANCER RESEARCH 40, 3804-3809. October 1980] 0008-54 72 780 /0040-OOOOS02.00 Elevated Activities and Properties of A and B and B- Variant in Human Lung Tumors1

Shinsei Gasa, Akira Makita, Toru Kameya, Tetsuro Kodama, Eiji Araki, Takeshi Yoneyama, Motohiro Hirama, and Masato Hashimoto

Biochemistry Laboratory. Cancer Institute ¡S G.. A. M.I. Department ol pathology IM. H.I. and Department ol Surgery ¡Ma.H.I. Hokkaido University School ol Medicine. Sapporo 060. and Pathology Division. National Cancer Center Institute [To. K , Te. K.I. Biochemical Laboratory ¡E.A.I. and Department of Surgery ¡T.Y.I. National Cancer Center Hospital. Chuo-ku. Tokyo 104. Japan

ABSTRACT increases of sulfated complex carbohydrates in lung carcinoma could result from diminished activity of arylsulfatases. Reduc The activities of arylsulfatases A and B were determined in tions of these occur with hereditary sulfatidosis (for human primary and secondary tumor tissues (total, 53 cases) a review, see Ref. 6) and (for a review, of various histológica! types. Significantly higher activities of see Ref. 14). Contrary to expectation, the activities of arylsul these were found in almost all the primary lung fatases A and B in lung neoplasm were significantly higher than carcinomas as compared to their corresponding uninvolved those in normal tissue from the same patients. Furthermore, tissues. No significant correlation was demonstrated between separation of arylsulfatases from the neoplasm revealed an the activities and histological figures (stroma amounts, additional component other than arylsulfatases A and B. etc.). Lung adenocarcinoma and squamous cell carcinoma showed the presence of an additional component (Bt) MATERIALS AND METHODS which was not detected in normal human lung. The tumor Chemicals. Potassium salts of p-nitrocatechol sulfate and p- arylsulfatase Bi had an isoelectric point (pi) of 6.7 and was clearly distinguished from (pi 4.9) and arylsul nitrophenyl sulfate were purchased from Nakarai Chemical Co., Kyoto, Japan; 4-methyl umbelliferyl sulfate was from Eastman fatase B (pi 9.1 to 9.2) in normal lung and lung tumor. The Kodak Co., Rochester, N. Y.; u-ascorbic acid 2-sulfate was tumor B, enzyme was demonstrated to be most probably an isoenzyme of arylsulfatase B, since this unusual enzyme was from Sigma Chemical Co., St. Louis, Mo. Other chemicals were indistinguishable from arylsulfatase B in terms of Ag + inhibition; of analytical grade. UDP-W-acetylgalactosamine 4-sulfate, and its kinetic parameters of Km for p-nitrocatechol sulfate, which UDP-A/-acetylgalactosamine were generously donated by Dr. was 2.9 rriM with Bi¡optimum pH of 6.3 for B,; heat stability; Sakaru Suzuki, Faculty of Science, Nagoya University, Nagoya, and substrate specificity for three synthetic and two physiolog Japan, and Dr. Akira Yoshida, Department of Biochemical ical substrates. Genetics, City of Hope National Medical Center, Duarte, Calif., respectively. L-Tyrosine O-sulfate was synthesized by the method of Tallan et al. (24) as modified by Dodgson et al. (4). INTRODUCTION Tumors. Human lung tissue obtained at surgery was quickly Three types of arylsulfatases, A, B, and C (arylsulfate-sulfo- separated into tumor which was macroscopically nonnecrotic , EC 3.1.6.1), are distinguished from one another in and the macroscopically normal portions, and it was stored immediately at —80°until use. All the tissues were subjected terms of substrate specificity, response to inorganic inhibitors, pH optimum, subcellular localization, and solubility (for review to histopathological examination and characterized as de of these sulfatases, for example, see Ref. 21). Arylsulfatases scribed previously (29). Pathological characterizations in A and B are localized mainly in lysosomes and can be easily cluded histological type, degree of differentiation, necrosis, obtained in a soluble form, while arylsulfatase C is present stroma, and inflammation. When inflammation was observed, mainly in microsomal membranes. Arylsulfatase A hydrolyzes the tissue frequently was infiltrated with lymphocytes and, in a the sulfate ester of cerebroside sulfate (15) and ascorbic acid few cases, with plasma cells, macrophages, or neutrophils. 2-sulfate (20), while arylsulfatase B acts on A/-acetylgalacto- The enzyme levels were examined in relation to histological samine 4-sulfate (10, 25). findings (Chart 2). Increased activities of arylsulfatases A and B in urine of Preparation of Enzymes. All operations were carried out at 2-4°. The tissue was homogenized for 3 min with 9 volumes of bladder tumor patients (3, 19) and arylsulfatase B in colorectal carcinomas (17) have been documented. The activity of aryl 0.25 M sucrose by a Brinkmann homogenizer followed by sulfatases was either elevated or depressed in a tissue-specific centrifugation at 500 x g for 10 min. The supernatant was manner in visceral neoplasms (7, 16). directly assayed for arylsulfatase activity or subjected to the We previously demonstrated increased enzyme separation. The 500 x g supernatant was sonicated in human lung carcinomas of 3 histological types (11, 12) and twice for 1 min with a Kontes sonifier and centrifuged for 20 an elevated level of cerebroside sulfate in lung adenocarcinoma min at 20,000 x g. The supernatant obtained was adjusted to (29). The present study was undertaken to examine if these a final concentration of 50% by the addition of crystalline ammonium sulfate. After centrifugation, the precipitate was 1Supported by Grants-in-Aid for Cancer Research (1979) from the Ministry of dialyzed against 10 HIMTris-HCI, pH 8.0, overnight. The dialy- Education. Science and nulture. and from the Ministry of Health and Welfare. sate was separated into arylsulfatases A and B on a DEAE- Japan. Received November 27, 1979; accepted July 9. 1980. cellulose (DE-52; Whatman Co.) column equilibrated previously

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. Arylsulfatases m Human Lung Tumors with 10 HIMTris-HCI, pH 8.O. Fractionation was also performed Tumors and Normal Lung. In Chart 1, arylsulfatase activities with a Model HLC-803 high-speed liquid Chromatograph (Toyo in various human lung tumors (classified into histological types) Soda Manufacturing Co., Tokyo, Japan) equipped with an IEX- are compared with values in the corresponding control lungs. 540DEAE column (1 5 cm x 7.5 mm, inside diameter) packed The arylsulfatase A-specific method was highly specific for with DEAE-2000SW (a DEAE derivative coupled with a syn arylsulfatase A, while the arylsulfatase B-specific method was thetic polymer carrier) and a 280 nm UV moniter. The pressure less specific, as has been described previously (1). Arylsulfa applied to the column was 50 kg/sq cm, and the flow rate was tase B activity which was determined on an unfractionated 60 ml/hr. Arylsulfatase B was eluted with 10 mw Tris-HCI enzyme specimen, therefore, might include some arylsulfatase buffer, pH 8.0, and then a density gradient from 0 to 0.5 M A activity. NaCI in the same buffer was applied to obtain a more acidic Although the activity varied between tumors, the arylsulfa form. An aliquot from 3-ml fractions was assayed for arylsul- tase A and B activities in all the tumors examined were elevated fatase activity. Similar separation was achieved by either when compared to those in the normal tissues from the same DEAE-cellulose or IEX-540DEAE columns. Therefore, subse individuals irrespective of histological types, with the exception quent fractionations were performed with the latter column due of arylsulfatase A in Tumor 103 and arylsulfatase B in Tumor to its rapid processing. 510. The increased activity of arylsulfatase B showed parallel Isoelectrofocusing. The enzyme was electrofocused in a increases of arylsulfatase A activity with exception of some column (100 ml) containing 1% LKB Ampholine carrier solution adenocarcinomas. Among the benign lung tumors available, (pH 3.5 to 10) with a stepwise sucrose gradient at 500 V for hamartoma showed some elevation of both arylsulfatases A 50 hr according to the method of Vesterberg (26). Fractions of and B activities. The level of these enzymes in benign lung 2 ml were collected at a flow rate of 48 ml/hr and assayed for tumors did not appear significantly elevated compared to nor their arylsulfatase activity. The enzyme-containing peaks were mal lung tissue. The mean specific activities of arylsulfatases concentrated by ultrafiltration. A and B were highest in adenocarcinoma followed by squa- Enzyme Assay. Arylsulfatase A and B activities were as mous cell carcinoma (Table 1). Lung tumors which had metas- sayed separately according to the method of Baum ef al. (1) tasized to the (Tumors 101 and 122) showed the same with a slight modification. The incubation mixture contained 5 activity as did primary lung tumors. On the other hand, arylsul mM p-nitrocatechol sulfate; 0.25 mM pyrophosphate; 0.8 M fatase A activity in secondary lung tumors which metastasized NaCI; 2 mg Triton X-100; 0.25 M sodium acetate buffer, pH from other tissues to lung, such as in osteosarcoma, thymus 5.0; and enzyme in a final volume of 0.4 ml for arylsulfatase A. carcinoma, leiomyosarcoma, and rectal carcinoma, was much The incubation mixture contained 25 mM p-nitrocatechol sul lower than in lung adenocarcinoma and squamous cell carci fate; 5 mM barium acetate; 1 mg Triton X-100; 0.25 M sodium noma and was similar to control values in the latter 2 carcinoma acetate buffer, pH 6.0; and enzyme in a final volume of 0.2 ml groups. The activity level of the adjacent normal lung tissue for arylsulfatase B. The reaction mixtures were treated identi from cancer patients appears to be a real property of the cally, and the amount of p-nitrocatechol liberated was assayed normal lung, since the activities of arylsulfatases A and B from at 515 nm. One unit refers to 1 nmol of p-nitrocatechol liberated lung tissue of non-cancer patients were also low. per hr. An increased level of arylsulfatase activity is present in the The reaction was carried out according to the urine of the patients with bladder inflammation (19). Since method of Tsuji ef al. (25) when UDP-W-acetylgalactosamine tumor tissue might be inflamed, the arylsulfatase activity was 4-sulfate was the substrate. The reaction product was chro- examined with respect to inflammation and other histological matographed on Whatman No. 3MM paper with isobutyric acid: characteristics. The activity level of arylsulfatases A and B in 0.5 M NH4OH (5:3, v/v) as the solvent in a descending way. tumor and normal tissues could not be correlated with the The paper was scanned for using a Shimazu type degree of inflammation, differentiation, or stroma figures (Chart CS-910 chromatogram scanner (350 nm reference beam and 2). 270 nm absorption beam). The RFwas 0.21 for UDP-N-acetyl- Fractionation of Arylsulfatases by AniónExchange Agent. galactosamine and 0.15 for UDP-A/-acetylgalactosamine 4-sul The arylsulfatases from tissue samples were analyzed for their fate. This procedure provided linear quantitation of the sugar ionic properties by DEAE column chromatography. Arylsulfa nucleotides from 5 to 30 nmol. When other substrates were tases of normal human lung were separated into 2 components, used, different methods were used. Desulfationofp-nitrophenyl termed Peaks B and A as shown in Chart 3. The elution profile sulfate was according to the method of Dodgson et al. (5), that of the enzymes was consistent with that of arylsulfatases B and of 4-methyl umbelliferyl sulfate was according to the method of A, respectively (27). Peak B, which was not retained on the Kihara ef al. (18) and L-tyrosine O-sulfate was desulfated anión exchange agent, showed only arylsulfatase B activity according to the method of Fluharty ef al. (8). Protein was using the assay for arylsulfatase B and no activity using the determined by the method of Lowry ef al. (13) with bovine assay for arylsulfatase A and was not inhibited by Ag*. There albumin as a standard. fore, the enzyme present in Peak B possessed the character Heat Stability of the Enzyme. Enzyme-containing solutions istics of arylsulfatase B. Peak A, which was eluted by NaCI were heated in 10 mM Tris-HCI buffer, pH 8.0, for 10 min at gradient, had arylsulfatase A activity as well as some 'arylsul fatase B" activity. various temperatures and activity was assayed for arylsulfatase A in Peak A or for arylsulfatase B in Peaks B and Bi. On the other hand, the carcinoma tissues had an additional enzyme designated as Peak B, in Chart 3, b and c, in addition to arylsulfatases B and A. Although the amount of B, varied RESULTS from one tumor to another, this arylsulfatase component con Activities of Arylsulfatases A and B in Individual Lung sistently appeared in adenocarcinoma, squamous cell carci-

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. S. Gasa et al. Adenocarcinoma Table 1 Statistical analysis ol arylsulfase A and B activities in human lung tumors Normal(U/mgprotein) Carcinoma(U/rngprotein) A B 20 40 60 80 40 20 O 100 typeAdenocarcinomaControlSquamousHistológica! (units/mgprotein)42.5 (units/mgprotein)32.3 101U ±13.9 ±19.9 110 ±31.2 ±37.0 ±19.0 115 carcinomaControlUndifferentiatedcell ±10.0 ±26.5 ±47.7.15.2,42.1,29.928.5,3.6"3.53.3e3.535.634.19.635.0(13)(12)(11)(12)( 119 carcinomaControlBenign ±26.1,17.2 4)( 2)( 120 2)( 2)( 121 tumorsControlSecondary ±30.88.7 4)( 2)( 202 1)( 1)( 4)Arylsulfatase 2) 205 tumorsmetastasized ±8.1a'3.74.2"4.12.02091.03.0b(23f04)(18)(17)( to lungArylsulfatase 207 209 Mean ±S.E. 6 p < 0.05 (f test; lung adenocarcinoma versus the corresponding normal 304 tissue). 305 c Numbers in parentheses, number of tumors. 306 d p < 0.001 (( test; lung squamous cell carcinoma versus the corresponding 403 normal tissue). 405 e p < 0.01 (f test; lung squamous cell carcinoma versus the corresponding 407 normal tissue). 410 411 414 501 503 50- C 504 509 O ,0

a. O0o9Pe-8oIn'normalAa 1Strom'O*.A-'•i+++Necrosis«if*p.•;«Ml*•••WDifferential Undifferentiated Carcinoma 1"••••i*animation„«i*":••D ? » Nomai (U/mgprotein) Carcinoma(U/mqoroteln) 40 20 O 20 40 60 80 100

206L 406L 203S 413S Squamous Cell Carcinoma ionIinor Normal (U/mg oroteln) Carcinoma (U/mg protein) tO 20 n 20 no §0 80 JIJO 103 Chart 2. Arylsulfatase B specific activity (ordinate) with respect to histological 106 figures. P. poorly differentiated carcinoma; M, moderately well-differentiated 109 carcinoma; W, well-differentiated carcinoma. •,squamous cell carcinoma; • 117 adenocarcinoma; A. undifferentiated carcinoma; CH,benign tumor; —,+. + + , 122° + + +, extent; <), normal tissue. 301 302 noma, and undifferentiated lung carcinoma (large cell carci 303 noma). The additional arylsulfatase of lung tumors was eluted 101 102 when the ionic strength in the buffer was increased by NaCI Hirt and possessed only arylsulfatase B activity. If component B, 408 has properties similar to those of arylsulfatase B, the activity is 409 415 much greater than shown in Chart 3, b and c, since arylsulfa 502 tase B activity is inhibited by NaCI. When 0.5 mivi AgNO3, an 510 arylsulfatase A inhibitor (21 ), was present in the assay system, 512 513 Peak B and B, enzymes were unaffected while the activity of Peak A enzyme was inhibited approximately 75%. Benign Tumors Isoelectric Focusing of Arylsulfatases. Peak A enzyme from Normal (U/mg protein) Timor (U/moprotein) 0 20 o o 20 40 60 80 100 normal lung and adenocarcinoma tissues demonstrated the same isoelectric point (pi) of 4.9 as shown in Chart 4. Peak B 111 201 412 Chart 1. Specific activities of arylsulfatases A and B in individual lung tumors 506 and normal lung. Values on the right, enzyme activities of individual tumors; values on the left, activities of their corresponding normal tissues; middle column, Secondary Tumors individual patient number; , arylsulfatase A-specific activity, . arylsul Normal(U/mgprotein) Carcinoma (U/TO protein) fatase B-specific activity. •,notdetermined because of lack of material; ", lung 40 20 n 0 20 40 60 80 100 tumors which metastasized to liver In undiflerentiated carcinomas. L indicates large-cell carcinoma, and S indicates small-cell carcinoma In other primary 204 208 tumors are Nos. 111 and 412. hamartoma; No. 201, fibromatosis; No. 506. 507 adenoma. Secondary tumors which metastasized to lung from tumors of other tissues are No. 204, osteosarcoma; No. 208, thymus carcinoma; No. 507, rectum 508 adenocarcinoma; No. 508, leiomyosarcoma.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. Arylsulfatases in Human Lung Tumors fatase was rapidly inactivated at temperatures above 45°,while Peak BI as well as Peak B arylsulfatase were relatively stable at temperatures below 55°(Chart 7).

1.0- 05 Substrate Specificity of Arylsulfatases. The 3 arylsulfatase i ¿ components of lung tumor tissue had the same specific activity O with 4-methyl umbelliferyl sulfate as a substrate (Table 2). < < Tyrosine sulfate and ascorbic acid 2-sulfate, known substrates 0.5 oc for arylsulfatase A, were cleaved only by the enzyme in Peak A. UDP-N-acetylgalactosamine 4-sulfate, a known arylsulfatase B substrate, was cleaved preferentially by Peak B and B, 20 ¿0 60 80 100 enzymes. These confirm the assignment of arylsulfatase A to FRACTION NUMBER Peak A and arylsulfatase B to Peaks B and B,. On the basis of the results, it seems that Peak B, arylsulfa 10l 0.5 tase component, present in human lung tumor but absent from u) O normal lung, is most probably a variant form of arylsulfatase B.

These results demonstrate that the arylsulfatases of lung carcinoma consist of 3 components in contrast to 2 compo 00 nents in normal human lung tissue, which is consistent with 10 20 30 FRACTION NUMBER previous observations on normal lung (27). The additional component present in the tumors was demonstrated probably to be a variant of arylsulfatase B. Multiple forms of arylsulfatase B have been noted in ox brain (2), human skin fibroblasts (22),

05 and human brain (23). Wasserman and Austen (28) demon strated that rat lung arylsulfatase B has a distinctly lower pi g (6.4) than that of the human lung. This lower value was obtained with cells possessing a single arylsulfatase as in eosinophils, B: oc or in cells that also contain arylsulfatase A as in mast cells and basophil leukemia tumor. Properties of rat arylsulfatase B (28) and a minor variant, termed Bm (pi 7.0), of human brain CO 10 20 30 arylsulfatase B (23) are very similar to the arylsulfatase B, of FRACTION NUMBER human lung tumor. It is conceivable that a portion of arylsulfa Chart 3. Anion-exchange chromatography of arylsulfatases. a, normal lung tase B present in normal lung is replaced by the B, enzyme using DE-52 column; b. adenocarcinoma using IEX-540DEAE columns; c, squa- mous cell carcinoma using IEX-540DEAE columns. Arylsulfatase A (O) and B due to intracellular control occurring during the course of lung (•)specific activities and protein ( ) are shown. tumor development. The present study demonstrated that the activities of aryl enzyme from either tissues had a pi of 9.1 to 9.2. The isoelectric sulfatases A and B were increased in almost all the cases of profile of the tumor Peak B, enzyme was pi 6.7 and was clearly lung carcinomas compared to the activities from the corre distinguished from Peak A and B enzymes. sponding control tissues. Morgan ef al. (16) reported similar Time Course Studies of Arylsulfatases. The time course of results with the mixed arylsulfatases in autopsied lung neo the reaction of arylsulfatase components obtained by DEAE plasm of undefined histological type. The elevated activities chromatography was studied using p-nitrocatechol sulfate as were statistically significant in the former 2 histological types. substrate (Chart 5). Peak A of arylsulfatase from adenocarci Although the number of cases of tumor and its control that noma showed a typical anomalous curve for arylsulfatase A, in were examined was not large, the activity of arylsulfatase A in which the reaction velocity is not strictly proportional to the benign lung tumors and secondary tumors metastasized to reaction time (21). Peaks Bt and B showed a linear curve for lung was nearly in the normal range. The activity of lung tumors the hydrolysis of the substrate. Arylsulfatases from squamous metastasized to liver was at the same level as that of primary cell carcinoma showed the same kinetics as did the enzymes lung tumors. Therefore, the elevation of arylsulfatase A seems from adenocarcinoma. Peak A, B, and Bi arylsulfatases from to be characteristic for lung carcinoma. Increased activity of adenocarcinoma had Km s for p-nitrocatechol sulfate of 0.3, arylsulfatases in lung tumor is probably due to a general 10.5, and 2.9 HIM and Vmax'sof 330, 4800, and 3140 units/ increase in lysosomal enzymes, because the activity of ft- mg protein, respectively. glucuronidase and /¿-N-acetylhexosaminidase was also in creased significantly in human lung tumor/' Normal and neo- pH Optimum of Arylsulfatases. The pH optimum of Peak A arylsulfatase from adenocarcinoma (Chart 6), squamous cell plastic tissues consist of heterogeneous cell populations and carcinoma, and normal lung was 5.2 to 5.4. The pH optimum the histological features in individual tissues vary from one to of Peak B displayed a relatively broad range with a peak near another which may result in a widespread variation of the 6.2. Peak B, arylsulfatase from carcinoma had a pH optimum enzyme activity values. However, since the activity values of of 6.3. Heat Stability Studies on Tumor Enzymes. Peak A arylsul M Narita. T. Mitsuyama. N. Taniguchi. and A Makita. unpublished data.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. S. Gasa ef al.

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\_Joo; "::_-_-;;;A,,-1.510H*•0.500,;>fi*"•'.51.00500 II A «0 10 20 30 FRACTION NUMBER FRACTION NUMBER Chart 4. Isoelectric focusing of arylsulfatases. a, enzyme preparation from normal lung isoelectrofocused prior to DEAE-chromatography. Peaks A in b, B in c, and Bi in d from lung adenocarcinoma were isoelectrofocused after being fractionated by DEAE chromatography.

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Chart 5. Time course of arylsulfatase activity. Activities of arylsulfatases from adenocarcinoma are determined with the arylsulfatase A-specific method for Peak A ( i. 85 units each) and with the arylsultatase B-specific method for Peak B (•.28 units each) and B, ( x . 20 units each) enzymes pH the arylsulfatases did not correlate directly with any of the Chart 6. Effect of varying pH on arylsulfatases. Activities of arylsulfatases histological changes concomitant with neoplasm, the altered from adenocarcinoma were determined by an arylsulfatase A-specific assay method for Peak A enzyme (.122 units each), and by an arylsulfatase B- enzyme levels in the tumor tissues must indeed represent specific method for Peak B enzyme (•,27 units each) and Peak B. enzyme (x, neoplastic changes. 66 units each). Elevations of arylsulfatase activities, however, do not appear to be general for all neoplasms in different tissues. The activi breast cancer (7) in comparison to normal tissues, although the ties of mixed arylsulfatase in human liver and neoplasms activities in colon (7, 16, 17), stomach, and skin cancers (7) were decreased (16). Differences could not be observed in were increased.

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REFERENCES

1. Baum, H., Dodgson, K. S., and Spencer, B. The assay of arylsulphatases A and B in human urine. Clin. Chim. Acta, 4: 453-455, 1959. 2 Bleszynski. W S., and Roy. A B Some properties of the sulphatase B of ox brain Biochim. Biophys. Acta, 377 164-171, 1973 3 Boyland. E., Wallace, D. M., and Williams. D. C. The activity of the enzymes sulphatase and /¡-glucuronidase in the urine, serum and bladder. Br. J. Cancer. 9 62-79. 1955. 4. Dodgson. K. S., Rose, F. A., and Tudball, N. Studies on sulphatases: 23. The enzymic desulphation of tyrosine O-sulphate. Biochem. J.. 71: 10-15. 1959 5 Dodgson. K S.. Spencer. B.. and Thomas. J Studies on sulphatases: 9 The arylsulphatases of mammalian . Biochem. J.. 59. 29-37, 1955. 6. Dulaney. J. T.. and Moser, J. W. Sulfatide lipidosis. In: J. B. Stanbury. J. B. Wyngarden. and D. S. Fredrickson (eds.). The Metabolic Basis of Inherited Disease, Ed. 4, pp. 770-809. New York: McGraw Hill Book Co.. 1978. 7. Dzialoszynski, L. M., Kroll, J. L., and Fröhlich. A. Arylsulphatase activity of some malignant tumors Clin. Chim. Acta. 14: 450-453. 1966. 8. Fluharty. A. L.. Stevens. R. L . Goldstein. E. B.. and Kihara. H The activity of arylsulfatase A and B on tyrosine O-sulfates. Biochim Biophys Acta. 566 321-326, 1979. 9. Gasa, S., Makita, A.. Hirama. M., and Kawabata. M. Cerebroside sulfotrans- ferase activity in human lung tissues: an elevated level in lung adenocarci 65 75 noma J Biochem. (Tokyo). 86 265-267. 1979. 10. Gorham. S. D.. and Cantz. M. Arylsulphatase B, an exosulphatase for TEMPERATURE (-c) chondroitin 4-sulphate tetrasaccharide. Z. Physiol Chem., 359 1811- 1814. 1978. Chart 7. Heat stability of arylsulfatases. The activity of Peak A enzyme (O, 11. Hatae, Y., Atsuta. T., and Makita, A. Glycosaminoglycans in human lung 126 units each) was determined using an arylsulfatase A-specific method, and carcinoma. Gann. 68. 59-63, 1977. those of Peak B enzyme (•.93 units each) and Peak B, enzyme (x, 125 units 12. Hatae. Y.. Yoda. Y., and Makita, A. Glycosaminoglycans in small cell each) were determined using an arylsulfatase B-specific method carcinoma of human lung: histologically characteristic pattern. Gann. 70. 389-390. 1979. Table 2 13. Lowry. O. H.. Rosebrough, N. J.. Farr. A L . and Randall, R J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193. 265-275. Substrate specificity of arylsulfatases from adenocarcinoma 1951. Enzyme activity (nmol/hr/ 14. McKusick. V. A., Neufeld, E. F., and Kelly, T. E. The mucopolysaccharide mg protein) storage diseases. In J. B. Stanbury. J. B. Wyngarden. and D. S. Fredrickson (eds.). The Metabolic Basis of Inherited Disease. Ed 4. pp. 1282-1307. Substrates B, New York: McGraw Hill Book Co.. 1978. sulfatep-Nitrocatechol4-Methylumbelliferyl 15. Mehl. E . and Jatzkewitz. H. Cerebroside 3-sulfate as a physiological sub sulfateArylsulfatase strate of arylsulfatase A. Biochim. Biophys. Acta. 151. 619-627. 1968. specificArylsulfataseA 16. Morgan. L. R.. Reehlmann. N. J.. Maddux, B., Rodriguez. L., Gidman, G., specificp-NitrophenylB Samuels. M. S.. and Krementz, E. T. Arylsulphatase activity of human lung, sulfateL-Tyrosine liver and kidney neoplasms. Clin. Chim. Acta. 32 316-320. 1972 O-sulfateUDP-W-acetylgalactosamine 17. Morgan, L. R.. Samuels, M. S., Thomas, W., Krementz. E. T., and Meeker. 4-sulfateAscorbic W. Arylsulfatase B in Cancer (Phila.). 36. 2337-2345. acid 2-sulfate3811552225791112138613044902703850199920280 1975. 18. Porter, M. T.. Fluharty, A. L.. and Kihara. H. Metachromatic leukodystrophy: arylsulfatase-A deficiency in skin fibroblast cultures. Proc. Nati. Acad. Sci. U. S. A.. 62 887-891, 1969 Our previous studies demonstrated an increase of cerebro- 19. Posey, L. E.. and Morgan. L R. Urine enzyme activities in patients with side sulfate content in human lung adenocarcinoma (29) and transitional cell carcinoma of the bladder. Clin. Chim. Acta. 74: 7-10. 1977. 20. Roy. A. B L-Ascorbic acid 2-sulphate: a substrate for mammalian arylsul marked elevations of glycosaminoglycans in lung carcinomas phatases Biochim. Biophys. Acta. 377 356-363, 1975 of 3 histological types (11, 12). In this study, however, the 21. Roy. A. B., and Trudinger. P. A. The sulphatases. In The Biochemistry of activity of arylsulfatase A, which acts on Cerebroside sulfate, Inorganic Compounds of Sulphur, pp. 133-189. Cambridge: Cambridge University Press, 1970. was increased not only in adenocarcinoma but also in other 22. Stevens. R. L. Minor anionic arylsulfatases in cultured human fibroblasts. histological types, being not significantly different between Biochim. Biophys. Acta, 370 249-256. 1974. these histological types of carcinomas. Therefore, it is probable 23 Stevens. R L.. Fluharty. A. L . Killgrove. A R.. and Kihara. H Arylsulfatases of human tissue: studies on a form of arylsulfatase B found predominantly in that the elevation of Cerebroside sulfate in adenocarcinoma is brain Biochim. Biophys. Acta. 481: 549-560. 1977. due to the increased activity of its synthetic enzyme, cerebro- 24. Tallan, H. H., Bella. S. T.. Stein. W. H., and Moore, S. Tyrosine O-sulfate as a constituent of normal human urine. J. Biol. Chem., 2 17 703-708, 1955 side (9), but not due to the diminished activity 25. Tsuji. M.. Hamano, M , Nakanishi, Y , Ishihara. K . and Suzuki, S. The of its degradative enzyme, arylsulfatase A. Similarly, it is con enzymatic desulfation of sulfated sugar nucleotides by a novel sulfatase in ceivable that the increased concentrations of glycosaminogly- hen oviduct. J. Biol Chem.. 249. 879-885. 1974. 26 Vesterberg. O. Synthesis and isoelectric fractionation of carrier ampholites. can (chondroitin 4-sulfate and ) may result Acta Chem. Scand.. 23 2653-2666. 1969 from the enhanced activity of one or more enzymes involved in 27. Wasserman, S. I., and Austen. K. F. Arylsulfatase B of human lung: isolation, synthesis, rather than from a reduction of characterization, and interaction with slow-reacting substance of anaphy- laxis. J. Clin. Invest , 57. 738-744, 1976. the activity in a degradative enzyme, arylsulfatase B. 28. Wasserman, S. I , and Austen. K. F Identification and characterization of arylsulfatase A and B of the rat basophil leukemia tumor J Biol Chem . ACKNOWLEDGMENTS 252. 7074-7080, 1977. 29. Yoda, Y., Gasa. S., Makita. A., Fujioka. Y., Kikuchi, Y., and Hashimoto. M. We are grateful to Drs. Shichiro Ishikawa and Yukio Shimosato (National Glycolipids in human lung carcinoma of histologically different types. J. Nati. Cancer Center. Tokyo) for their encouragement and supply of the tumors. Cancer Inst.. 63 11 53-1160, 1979.

OCTOBER 1980 3809

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. Elevated Activities and Properties of Arylsulfatases A and B and B-Variant in Human Lung Tumors

Shinsei Gasa, Akira Makita, Toru Kameya, et al.

Cancer Res 1980;40:3804-3809.

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