278 Biochimi¢~ etBiophysicaAeta 888 (1986) 278 281 Elsevier

BBA 11856

Sial ic acid lyase in human promyelocyt ic leukemic cel ls (HL-60) during

phorbol-ester- induced differentiat ion

L e o n a r d W a r r e n

The Wistar Institute of Anatomy and Biologo', 36th Street at Spruce, Philadelphia, PA 19104 (U.S.A.)

(Received 12 May, 1986)

Key words: Sialic acid lyase; Tumor promoter; Differentiation; (Leukemia cell)

There is a marked increase in the activity of sialic acid lyase (N-acetylneuraminate lyase; EC 4.1.3.3; also known as sialic acid aldolase) in HL-60 cells induced to differentiate into macrophages by the phorbol ester, tetradecanoylphorbol 12-myristate 13 acetate (TPA). Exposure of HL-60 cells to retinoic acid, butyric acid or dimethyl sulfoxide has little or no effect. The level of the enzyme remains unaltered in HL-60 cells grown in the presence of an inactive analog of TPA, nor does it change in variants of HL-60 cells resistant to TPA.

Introduction

Extensive changes take place in the bound carbohydrate groups of HL-60 cells when these cells are induced to differentiate by exposure to the phorbol ester, TPA. [1] In studies designed to explain the enzymatic basis of the changes, it was found that the level of cellular N-acetylneurami- hate lyase increased markedly as has been found when mouse tetratocarcinoma cells differentiate spontaneously or are induced to differentiate by re t inoic acid and o ther agents [2]. N- Acetylneuraminate lyase, found in bacteria [3-8] and animal tissues [9-12] cleaves free N- acetylneuraminic acid into pyruvate and N-acetyl- D-mannosamine. Although the reaction is reversi- ble, cleavage of N-acetylneuraminate is favored so that at equilibrium 90% of N-acetylneuraminate is destroyed [9]. The precise role of the enzyme in sialic acid metabolism is not really known, but its

Correspondence address: The Wistar Institute of Anatomy and Biology, 36th Street at Spruce. Philadelphia, PA 19104, U.S.A. Abbreviations: DMSO, dimethylsulfoxide; TPA, tetrade- canoylphorbol 12-myristate 13-acetate; ManNAc, N-acetyl-o- mannosamine; 4a-PDD, 4c~-phorbol 12,13-didecanoate.

equilibrium would suggest that it could provide cells with pyruvate and N-acetylhexosamine from exogenous sialic acid. This notion is strengthened by the common association of neuraminidase and N-acetylneuraminate lyase, e.g., in Vibrio cholerae and Clostridium perfringens. Since N-acetylneura- minate is almost always in a bound state, neu- raminidase would be necessary to convert it to a free form that is susceptible to N-acetylneurami- hate lyase.

In this paper are described some characteristics of the enzyme N-acetylneuraminate lyase during the differentiation process of HL-60 cells.

Materials and Methods

HL-60 cells [13] were obtained from R.C. Gallo and a line of these cells, R-l , resistant to TPA [14] was kindly provided by L. Diamond. Cells were cultured in T150 Falcon flasks in RPMI-1640 tissue culture medium supplemented with 10% fetal calf serum. TPA was added to cultures at a concentration of 5 . 1 0 - s M. Differentiation was indicated by cell attachment and spreading [15]. After 24 or 48 h of culture, the medium, with unattached cells, was poured off. Attached cells

0167-4889/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

were washed twice with phosphate-buffered saline, removed by scraping and pelleted by centrifuga- tion. An equal volume of 0.1 M potassium phos- phate buffer (pH 8.0) was added to the pellet, which was homogenized by ten vigorous strokes in a Potter-Elvejhem homogenizer (5 ml) with a Teflon pestle. The homogenate usually contained 15-20 mg protein per ml.

N-Acetylneuraminate lyase was assayed by in- cubating N-acetyl[14C]neuraminic acid and 100- 500/~g enzyme protein in a final volume of 30/L1 containing 20/~mol sodium phosphate buffer (pH 8.0). The specific activity of the N-acetylneurami- nate, (N-acetyl[4-9-t4C]neuraminic acid) was ad- justed so that there were 50 nmol N-acetylneura- minate containing 0.05 /~Ci X4C in each vessel. After 2 h of incubation at 37°C, 20 /~1 of the incubation mixture were spotted on Whatman 3MM paper, which was subjected to high-voltage electrophoresis (2500 V) in 0.1 M pyridine acetate buffer (pH 5.6) for 90 min in a Gilson high-volt- age electrophoresis apparatus (Model D). A 4 cm ~ area at the origin for each vessel, where the radio- active product of the reaction, N-acetyl-D-man- nosamine, remained, was cut out, immersed in 4 ml Formula 963 NEN scintillation fluid. Radioac- tivity was determined on an Intertechnique Liquid Scintillation Counter SL300.

Results

Under the conditions of the assay, the forma- tion of N-acetyl-D-mannosamine from N-acetyl- neuraminate is linear with enzyme concentration and with time for 5 h of incubation. The optimal pH of the reaction is 8.0 and the K m of N-acetyl- neuraminate is 1-2 mM. At least 90% of the enzyme activity could be recovered in the super- natant after centrifugation of the cell homogenate at 100000 × g for 1 h. It was found that as N-acetylneuraminate is destroyed, as measured by the thiobarbituric acid assay [16], equimolar amounts of pyruvate and ManNAc are formed. Pyruvate was measured by the decline in absorp- tion of NADH (340 mm) in the presence of lactic acid dehydrogenase [4]. ManNAc was measured by the assay of Reissig et al. [17]. Both pyruvate and ManNAc products chromatograph with authentic standards in two solvent systems on

279

141

12

J~

10

0 0 < 8 8 Z c ~

E

o E 4 2

-

I I I 0 24 48

Exposure time to TPA (hrs)

Fig. 1. Increase in N-acetylneuraminate lyase activity in HL-60 cells exposed to TPA (5-10 ~ M). Differentiated, adherent cells and nondifferentiated, nonadherent cells were assayed separately. Assay as described in text.

Whatman 3MM paper before and after impreg- nation with sodium borate [18] as described in Ref. 2. The products were detected by a silver nitrate procedure [19]. The reduced and acetylated ManNAc product form a single peak with authentic ManNAc when subjected to gas chro- matography [20].

Upon addition of TPA to a culture of HL-60 cells, most but not all of the cells differentiate and become adherent and spread on the surface of the plastic flask (Falcon) in which they are growing. At 24 and 48 h, adherent and nonadherent cells were harvested and assayed for enzyme activity.

TABLE I

N-ACETYLNEURAMINATE ACTIVITY IN HL-60 CELLS

Assay is described in text. Cells were exposed to the inducers for 48 h.

Inducer Concentration nmol (M) N-acetylman-

nosamine formed

None 4.50 TPA (nonadherent cells) 5-10 ? 4.80 TPA (adherent cells) 5-10 7 16.80 Retinoic acid 1 - 10 6 3.80 Butyric acid 1-10 4 5.75 DMSO 0.14 3.05 4a-PDD 1.6-10 6 4.10

280

TABLE II

ENZYME ACTIVITY IN HL-60 CELLS

TPA, 4-10 -7 M, 24 h. The assay is described in the text.

n m o l / m g protein per h

Control cell 1.5 TPA-non adherent 2.5 TPA-adherent 13.4 TPA-adherent + cycloheximide (10/2g/ml) 3.4 TPA-adherent + puromycin (30/Lg/ml) 2.9

Fig. 1 shows that after 48 h, activity rose 4-5-fold in the adherent, differentiated cells but declined slightly in the cells remaining in suspension.

Exposure of HL-60 cells to an inactive analog of TPA, 4a-phorbol 12,13-didecanoate (4~x-PDD), ( 5 " 10 8, 5 " 10 - 7 M ) has no effect on the mor- phology of the cell or the level of NAN-lyase activity (Table I) nor does TPA alter the level of enzyme activity in R-l , a TPA-resistant line of HL-60 cells [14]. TPA added to the assay vessel at concentrations of 5 . 1 0 s and 5 . 1 0 7 M has no effect on the reaction.

TABLE III

ENZYME ACTIVITIES IN U N D I F F E R E N T I A T E D AND D I F F E R E N T I A T E D CELLS

Activity is expressed as pmol N-acetylneuraminate trans- f e r red /mg protein per h. Assays were carried out essentially as previously described [19]. Incubation vessels contained 0.10 gCi (2 nmol) cytidine monophosphate N-acetylneuraminate [4 9-14C]CMPNAN, 50 p~g acceptor, 5 p, mol potassium phos- phate buffer (pH 7.0) 10 3 M MgCI 2, and Triton X-100, 0.1% final concentration in a volume of 50 ~1. After 2 h of incuba- tion the vessel contents were spotted on 3MM paper and subjected to high-voltage electrophoresis as described. Prod- ucts were located with a Vangard paper strip scanner. Radioac- tive areas were cut out and quanti tated as described for the N-acetylneuraminate-lyase assay. Antifreeze glycoprotein from T. borchgrevinki (disaccharide sidechains, D-Gal-N-acetyl-D- galactosamine) was the gift of Dr. A.L. DeVries. The experi- ment was done three times with essentially the same results.

Cell N-Acetyl- Sialyl transferase

neuraminate Antifreeze Asialofetuin lyase glycoprotein glycopeptide

acceptor acceptor

HL-60 1.3 17.4 18.7 HL-60 TPA 40.7 16.6 12.8

The data in Table I show that TPA, which induces cells to differentiate into macrophages [15], is the only agent tested that causes an eleva- tion of N-acetylneuraminate lyase activity. Reti- noic acid, butyric acid and dimethylsulfoxide, which induce myeloid differentiation [2], do not elevate the level of N-acetylneuraminate lyase. The increase in enzyme activity induced by TPA can be prevented by inhibitors of protein synthesis in the culture medium (Table II). Enzyme could not be detected in the medium of undifferentiated or differentiated HL-60 cells.

In Table III, it can be seen that, while TPA induces a marked elevation of N-acetylneurami- nate lyase in HL-60 cells, other enzyme activities in the very same cells, involved with the metabo- lism of sialic acid such as N-acetylneuraminate transferases change little, if at all.

Discussion

Previous work has shown that as HL-60 cells differentiate into macrophages, a radical decrease in synthesis of high-molecular-weight glyco- peptides takes place [1] accompanied by changes in the composition of glycolipids [22]. The glyco- peptide changes do not occur when HL-60 cell are induced by retinoic acid or DMSO to differentiate into a granulocytic type of cell. Other changes associated with bound carbohydrates have been described in differentiating HE-60 cells. Myeloid differentiation induced by agents such as retinoic acid or DMSO is accompanied by a 2.5- to 2.7-fold increase in sialidase activity [23], marked changes in the array of glycoproteins at the cell surface [24], and increases in the levels of activity of specific galactosyl, fucosyl and sialyl transferases, a second fucosyl transferase decreases in activity [25].

A different pattern of changes in transferase levels is seen in TPA-induced differentiation into macrophages. We have found little change in sialyl transferases. On the other hand, N-acetylneurami- nate-lyase activity increases in a cell induced to become a macrophage and this is consistent with the notion that a housekeeping cell such as the macrophage would require enhanced levels of N- acetylneuraminate lyase, a catabolic scavenging enzyme. The rationale for the modulation of N-ac-

e t y l n e u r a m i n a t e - l y a s e ac t iv i ty m a y h a v e l i t t le to

d o wi th tha t for o t h e r e n z y m e s i n v o l v e d in sial ic

ac id m e t a b o l i s m , w h i c h are b i o s y n t h e t i c in na tu re .

It appea r s tha t i n d u c e d d i f f e r e n t i a t i o n o f H L - 6 0

cel ls a l ong two s e p a r a t e pa th s is a s soc ia t ed wi th

two specif ic , d i sc re te sets of p h e n o t y p i c changes

i n v o l v i n g b o u n d c a r b o h y d r a t e s . I t is poss ib le tha t

the changes in b o u n d c a r b o h y d r a t e s o b s e r v e d in

H L - 6 0 cel ls as they d i f f e r en t i a t e i n to m a c r o p h a g e

c o u l d unde r l i e the p r o g r a m m e d a p p e a r a n c e of an-

t igens [26,27] a n d the d r a m a t i c inc rease in ad-

hes iveness in these cells.

Acknowledgements

I wish to t h a n k Ms. S. Pancraz i , R. Esp i r i tu

a n d T. B o g a n for exce l l en t t e chn ica l ass is tance .

T h i s w o r k was s u p p o r t e d by g ran t s C A 1 9 1 3 0 f r o m

the U.S. Pub l i c H e a l t h Serv ice and by R D P - 1 9 H

f r o m the A m e r i c a n C a n c e r Socie ty .

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