Polyadenylic acid polymerase activity in normal and leukemic human leukocytes

[CANCER RESEARCH 44, 3691-3697,

September 1984]

Polyadenylic Acid Polymerase Activity in Normal and Leukemic Human Leukocytes1 Theoni Trangas, Nelly Courtis, Gerassimos A. Pangalis, Helen V. Cosmides, Costas loannides, Mike Papamichail, and C. Milton Tsiapalis2 Departments of Biochemistry [T. T., N. C., C. M. T.] and Immunology (C. I., M. P.] of Papanikolaou Research Center of Oncology, 171, Alexandias Av., Athens 11522, Hematology Unit, First Department of Internal Medicine ¡G.A. P.], and Second Department of Pediatrics [H. V. C.], University of Athens, Athens 11522, Greece

ABSTRACT Soluble polyadenyljc acid [poly(A)] polymerase and poly(A) nucleases content of normal human blood lymphocytes and leukemic blood cell populations was determined. Blood lympho cytes from seven normal individuals were used as controls. Leukemic cells were obtained from 69 patients with various types of acute and chronic leukemias. Chronic lymphocytic leukemias presented poly(A) polymerase values with a mean of 9 ±4 (S.D.). Although most of the chronic lymphocytic leukemia cases presented poly(A) polymerase activities similar to those of normal lymphocytes (3 ±3), a small number fell into the specific activity values of acute leukemias, which were significantly higher and covered a wider range. The mean values for acute myeloblastic, acute monoblastic, and acute lymphoblastic leukemias were 53 ±50, 21 ±8, and 29 ±14, respectively. A statistically significant difference was found between chronic and acute leukemias (p< 0.01). The observed differences in poly(A) polymerase levels of acute lymphoblastic leukemia versus chronic lymphocytic leukemia per sisted after fractionation of the crude extracts and, furthermore, they could not be attributed to differences in the levels of poly(A)degrading enzymes Ipoly (A) endo- and exonucleases]. Fraction ation of leukemic extracts on Sephadex G-75 revealed two molecular forms of poly(A) polymerase activity. INTRODUCTION Poly(A)3 metabolism enzymes mediate adenylation and deadenylation of eukaryotic heterogeneous RNA and mRNA (12). These enzymes have been detected in several tissues, and some of them have been highly purified and characterized (12,13,18, 19, 25, 26). Several approaches have also been undertaken to elucidate the biological role of poly(A) metabolism enzymes in the regulation of eukaryotic gene expression (5, 8-12, 17, 20, 21,29,30). Most of them deal with measurements of the enzyme activities of cells in various physiological or pathophysiological conditions and in response to external stimuli. These studies have not produced a coherent pattern of the regulation of poly(A) metabolism enzymes. This is not surprising, in view of the 1 Presented at the Tenth International Symposium on Comparative Research of Leukemia and Related Diseases, August 31 to September 4, 1981, UCLA, Los Angeles, CA, and North Atlantic Treaty Organization International Advanced Study Institute on Biochemical and Biological Markers of Neoplastic Transformation, September 28 to October 8, 1981, Corfu, Greece (30). This work was supported by the Hellenic Anticancer Institute. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: ALL, acute lymphoblastic leukemia; AML. acute myeloblastic leukemia; CLL, chronic lymphocytic leukemia; PBL, peripheral blood lymphocytes; o4igo(A), oligomer of polyadenylic acid, chain length 10 residues; poly(A). polymer of polyadenylic acid, chain length more than 200 residues. Received June 5,1983; accepted May 31,1984.

SEPTEMBER

difficulty of measuring such enzyme activities in cell extracts (12, 13). Furthermore, such measurements should correlate with concomitant changes in the poly(A) metabolism of the cell. Among the various biological systems available for studying the biology of poly(A) metabolism enzymes, lymphocytes render themselves very attractive as is demonstrated in this report. Our objective was to investigate any putative differences in the levels of poly(A) metabolism enzymes among normal, ALL, AML, and CLL human blood leukocytes and to see possible correlations with respect to these differences and what is known about the patterns of poly(A) metabolism in lymphocytes (2-4,14, 24). MATERIALS AND METHODS Materials Tritium-labeled compounds were purchased from Amersham Interna tional-England; unlabeled nucleotides were obtained from BoehringerMannheim Tutzing, West Germany; GF/C and DE-81 sheets were from Whatman (Maidstone, Kent, England) products; Bio-Rad protein assay (dye reagent concentrate) was from Bio-Rad Laboratories (Richmond, CA); Sephadex G-50 and G-75 were from Pharmacia (Uppsala, Sweden); Bio-fluor was from New England Nuclear (Boston, MA); Folin-Ciocalteu's phenol reagent, ethanol, toluene, sodium chloride, mono- and dibasic potassium phosphate were from Ferak (Berlin, West Germany); diethylpyrocarbonate, urea, sucrose, bovine serum albumin, trichloroacetic acid, PRO, EDTA, and 2-mercaptoethanol were from Serva (Heidelberg, West Germany); POPOP, Trizma base, and sodium acetate were from Sigma Chemical Co. (St. Louis, MO); manganese chloride was from Merck (Darmstadt, West Germany); tetrasodium pyrophosphate was from BDH Biochemicals, Ltd. (Poole, England); RPM11640 medium and L-glutamine were obtained from Flow Laboratories (United Kingdom); and fetal calf serum was a product of Filtran (Filtran Pty, Ltd., Altona, Australia). Preparation of Soluble Cell Extracts and Protein Determination In 69 newly diagnosed leukemic cases, 20 ml of blood were collected in heparinized syringes before treatment. For the classification of our leukemic cases into various categories, conventional morphology (31), cytochemistry with myeloperoxidase, Sudan Black B and sodium fluo ride-sensitive naphthol AS -D acetate esterase (22), biochemistry with terminal deoxynucleotidyl transferase, and various immunological surface markers were performed. After the analysis of the above findings, 8 cases were classified as AML, 2 cases as acute monoblastic leukemia, 16 cases as ALL and 43 cases as B-CLL. Of the 16 ALL cases studied, 4 had a T-phenotype, and 12 were of the common cell type. Lymphocytes were isolated from peripheral blood by the Boyum (6). After 3 washes, cells were disrupted and protein by centrifugation over a layer of 9% sucrose containing according to the method of Berger and Cooper (3). A human

method of solubilized 1% NP-40 B-lympho-

blastoid cell line (Raji) was cultured in RPM11640 medium supplemented with 10% fetal calf serum in an incubator with 95% air and 5% COz at 37°at a density of 0.5 x 10°cells/ml. Cells were disrupted as described above. Protein determination was carried out by the method of Lowry ef al. (16) using albumin as standard unless otherwise specified.

1984

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3691

T. Trangasetal. RESULTS

Enzyme Assays Poly(A) Polymerase.

The assay measures the incorporation of (5'-8-

3H) ATP into acid-insoluble product using poly(A) or ougofA)™as initiators as described earlier (27, 28). The standard assay mixture (100 pi) contained 200 mM Tris MCI (pH 8.3), 1 mM MnCI2,1 mM [3H]ATP (20 to 30 cpm/pmol), 4 mM 2-mercaptoethanol, 1 mM (3'-OH) poly(A) or 0.2 mM oiigcHA). and 20 pi of the cell extract, diluted to a final concentration of 1 mg of protein per ml, to be assayed. After incubation at 37°,20-pl aliquots were spotted at various time intervals on GF/C discs and were processed and counted as described previously (27, 28). One unit of enzyme activity is defined as 1 nmol of radioactive ribonucleotide incor porated per hr. Specific activity is expressed as units of activity per mg of protein. Total Poly(A) Nucleases. The assay measures the conversion of high-molecular-weight radioactive poly(A) to low-molecular-weight prod ucts (18,27). The standard assay mixture (70 pi) contained 200 mM TrisHCI (pH 8.6), 1 mM MnCl;.. 4 mM 2-mercaptoethanol, 1 mM |:'H|poly(A) (1300 cpm/nmol). and 10 pi of the cell extracts, diluted to a final concentration of 1 mg of protein per ml, to be assayed. After incubation at 37°,for 0 and 10 min, 30-pl aliquots were placed on DE 81 discs. The discs were collected in 5 M urea, 2 M NaCI, and 5 mM EDTA. After washing 3 times (for 5 min each) with this solution, the discs were dried in pure ethanoi, scintillation fluid was added, and the discs were counted. One unit of enzyme activity is defined as the amount of enzyme which reduces, after incubation has taken place for 10 min, the radioactivity derived from [3H]-poly(A) and bound on DE 81 filters to 50%.

Poly(A) Polymerase Activity in Normal Human Blood Lym phocytes and Leukemic Cell Extracts. Addition of crude ex tracts to a standard reaction mixture resulted in the polymeriza tion of AMP. Under the conditions described in "Materials and Methods" this reaction followed linear Kinetics for up to 2 hr. No significant differences were observed between the poly(A)- and oligo(A)-initiated activities in either ALL (Chart 1A)- or CLL (Chart 16)-soluble cell extracts. Linearity was achieved through satu rating ATP levels (27) and a certain protein concentration. A relatively linear relationship existed between the rate of AMP polymerization and the concentration of soluble cell extract only within a range of 0.2 to 1.0 mg of protein per ml of the reaction mixture (Chart 2). The measurements of poly(A)-initiated activity of poly(A) polymerase in the soluble extracts of normal and leukemic cells from 76 individuals are presented in Chart 3. According to these results, the majority of the activity values of

CLL

•f, 80 Õ

Poly(A) Exonuclease. The assay contained the same components as the one described for the total poly(A) nudeases. After incubating the reaction mixture at 37°for 0 and 30 min, aliquots of 30 pi were placed on GF/C discs, and reaction products were precipitated with ice-cold 5% tnchloroacetic acid with 1% tetrasodium pyrophosphate. After 3 wash ings in 5% tnchloroacetic acid, the discs were dried in pure ethanoi and counted. One unit is the enzyme activity which releases 1 nmol of AMP in 30 min. Poly(U) Nucleases. The assay determines quantitatively the conver sion of high-molecular-weight [3H]poly(U) to taw-molecular-weight prod ucts. The standard assay mixture (50 pi) contained 100 mM sodium acetate (pH 4.7), 0.5 mM MnCl?. 0.05 mM 13H|poly(U) (30 cpm/pmol), and

Chart 1. ATP incorporation into polynucieotide as a function of time using crude cell extracts from lymphocytes of an ALL patient (A) and a CLL patient (B) with poiy( A) (O) and oligo( A) (•) as primers. The final protein concentration in the reaction mixture was 0.2 mg/ml.

25 pi of the cell extract, diluted to a final concentration of 1 mg of protein per ml. Incubation was carried out at 37°,and, at various time intervals, 10-»¡I aliquots were added to 10 pi of 0.5 HIM poiy(A). and the acid-

20

insoluble material of the reaction mixture was determined as in the poiy(A) exonuclease assay. One enzyme unit is defined as the amount of enzyme which releases 1 nmol of UMP in 30 min. Under these conditions, poiy(A) is not degraded (1). Gel Filtration of Poly(A) Nuclease Reaction Products Products of poiy(A) nudeases were chromatographed by the method of Müller(18). Reaction mixtures (0.2 ml) were chilled on ice and brought to a diethylpyrocarbonate concentration of 1 pi/ml and a 6 M urea final concentration. Samples were applied to a Sephadex G-50 column (0.9 x 30 cm), equilibrated, and ran with a buffer containing 50 mM Tris-HCI (pH 7.4) and 6 M urea at 4°.Twenty-drop fractions were collected, and their radioactivity

content was determined by counting 50-pl aliquots in

1 ml of Biofluor. Absorbance at 260 nm was also monitored.

ALL

8 1.5 21 ± 8 29 ±14 9± 4 3± 3

'p < 0.01 (comparing all acute leukemias versus CLL cases). 6 Mean ±S.D.

the CLL extracts was within the same range as those of normal lymphocytes. Only a small number of CLL cases (8 of 43) had markedly higher activity values. Blast cells from ALL and partic ularly from AML covered a wide range of activities, which, however, were above the bulk of CLL or normal ones. Table 1 presents the number of cases and the mean values of the enzyme activity per mg of protein for the leukemic cells of acute and chronic leukemias and the normal lymphocytes. The differ ences in the levels of this enzyme are statistically significant between acute and chronic leukemias (p < 0.01). On the basis of these results, blast cells from AML appeared to present a mean poly (A) polyrnerase activity value approximately 15-fold greater than that of normal blood lymphocytes, while blast cells from ALL presented a mean poly(A) polyrnerase activity as much as 3- and 9-fold greater as compared to lymphocytes from CLL and normal donors, respectively. To investigate the possibility that the presence of inhibitors or activators was responsible for the differences observed among the classes of leukocytic cells, mixing experiments were per formed. When lymphocytic extract from an ALL patient with poly(A) polyrnerase specific activity of 32 units/mg was added to an equal amount of a CLL lymphocytic extract with specific activity of 4 units/mg, the resulting specific activity was the mean of the separate values, i.e., 18 units/mg. Chart 4 shows the rates of the reactions of the above-mentioned ALL, CLL, and combined extracts. These results are typical of several experiments, in which all possible combinations of extracts were used within a group of 3 ALL, 2 AML, and 4 CLL cases. Sephadex G-75 Gel Filtration of Soluble Cell Extracts from SEPTEMBER

elution pattern between ALL and CLL extracts. All of the CLL enzyme (Chart 5A) was excluded on G-75, while only a small portion of the ALL enzyme (Chart 5ß)was excluded, and the bulk of the activity was retained and eluted as a smaller protein of M, 60,000. Additional work on the isolation and characteriza tion of the poly(A) polyrnerase from these 2 classes of leukemic cells is required to shed light on detailed differences in the molecular nature of the enzyme. Quantitative measurements of enzyme activities recovered from G-75 column chromatography are shown in Table 2. The examples included in this table fall into 2 categories in terms of poly(A) polyrnerase elution properties. The first category includes the ALL and AML cases whose elution pattern is illustrated in Chart 53; the second category includes the CLL and Raji cell line extracts with poly(A) polyrnerase that élûtes in the manner depicted in Chart 5A. Raji is a human leukemic cell line of Blymphocyte origin, as were all of the CLL cases examined, but has poly(A) polymerase activity higher than any CLL investigated in this study. Taking into account both poly(A) polymerase peaks eluting from Sephadex G-75, when present, the recovery of poly(A)-initiated activities was about 20% of the activity applied

I70

ALL

6001 -

50

040 ¡5

u E 30 CL

5 X> 10

Time

(hr)

Chart 4. Poly(A)-initiated, poly(A) polymerase activity in mixing experiments of ALL and CLL crude cell extracts. ATP incorporation into polynucleotide as a function of time in a standard poly(A) polymerase reaction mixture containing 100 ng of protein from an ALL crude cell extract (•),100