Leukemia Research Vol. 14, No. 3, pp. 273-278, 1990. Printed in Great Britain.

0145-2126/90 $3.00 + .00 Pergamon Press plc

P O L Y A D E N Y L I C ACID POLYMERASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA RYUHEI SASAKI,* JUN MINOWADA,'I FREDERICK J. BOLLUM:~ and YASUSADA MIURA* *Department of Hematology, Jichi Medical School, Tochigi, Japan; tHayashibara Institute, Okayama, Japan; SDepartment of Biochemistry, Uniformed Services, University of the Health Sciences, Bethesda, Maryland, U.S.A.

(Received 5 June 1989. Revision accepted 30 September 1989) Ahstract--Poly(A) polymerase activity was markedly elevated in CML in the blastic phase, moderately high in the accelerated phase and low in the chronic phase. The activity was significantly higher in the myeloid crisis than in the lymphoid crisis and elevated with increasing ratio of blasts in leukemia cases. In TPA or retinoic acid-treated leukemia cells poly(A) polymerase activity was decreased. These results suggest that poly(A) polymerase activity changes, depending on the maturation of leukemic cells and the assay of this enzyme activity may be useful for the early detection of the exacerbation of CML cases.

Key words: Polyadenylic acid polymerase, chronic myelogenous leukemia (CML) in chronic phase, accelerated phase, blastic phase, myeloid crisis, lymphoid crisis.

INTRODUCTION

(ALL) cases were assayed under the optimal conditions. The results show that the poly(A) polymerase activity changes, depending on the maturation of leukemic cells.

POLY(A) sequences are covalently linked to the 3' end of both the non-histone messenger R N A ( m R N A ) and heterogenous nuclear R N A of eukaryotes [2]. Poly(A) sequences may play a role in m R N A stability [6], in the transfer of m R N A from the nucleus to the cytoplasm [3], in the translational capacity of m R N A [3] and in cell differentiation [4, 11, 15]. Therefore, a change in the poly(A)metabolizing enzymes which regulate poly(A) sequences may indirectly modulate the protein synthesis. Recently, several studies on poly(A) polymerase activity in leukemic cells were reported [10, 11, 16]. However, there has been no investigation on this enzyme in chronic myelogenous leukemia (CML) cases. In this study, poly(A) polymerase activities in CML cases at various stages, in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia

MATERIALS AND METHODS

Reagents and clinical samples HL-60 cell line was maintained in RPMI-1640 containing 10% heat-inactivated fetal calf serum at 37°C in 5% CO2 and 95% air. The optimal conditions for poly(A) polymerase assay were determined using HL-60 cells. [2,83H]adenosine 5'-triphosphate (3H-ATP) and poly[83H]adenylic acid [3H-poly(A)] were bought from the Radiochemical Centre, Amersham, England. Poly(A), oligo A12_18, transfer RNA, calf thymus DNA and TPA (12-O-tetradecanoylphorbol-13-acetate) were from Pharmacia, P-L Biochemicals Inc., Milwaukee, WI. TPA was dissolved in 3.2 mM dimethyl sulfoxide. 13-cis-retinoic acid was obtained from Sigma Chemical Co., St Louis, MO and dissolved in 95% ethanol. Bone marrow aspirates or blood were obtained from leukemic patients, with their informed consent, separated with Ficoll-Hypaque, as described previously [13]. Subjects included 15 patients with CML in the chronic phase, 24 in the blastic phase, five in the accelerated phase, 14 AML and 14 ALL cases. None of the patients received cytotoxic drugs immediately before and at the time of this study. Blood was obtained from ten normal volunteers. The diagnosis of leukemia was based on conventional studies [5]. The blastic phase of CML was defined as 30% or more blasts in the bone marrow, the blood or both. The accelerated phase of CML was diagnosed according to the criteria described by Karanas and Silver [7].

Abbreviations: Poly(A), polyadenylic acid; m-RNA, messenger RNA; 3H-ATP, tritiated adenosine 5'-triphosphate; oligo A, oligoriboadenylic acid; AMP, adenosine monophosphate; TPA, 12-O-tetradecanoylphorbol13-acetate; Kin, Michaelis constant; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; TdT, terminal deoxynucleotidyl transferase. Correspondence to: Dr Ryuhei Sasaki, Department of Hematology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-Machi, Kawachi-Gnn, Tochigi, Japan, 329-04. 273

274

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FIG. 1. Poly(A) polymerase activities at various phases of CML cases. The enzyme activities are expressed as (A) units (n moles) of AMP polymerized per 5 x 1 0 6 cells and (B) units per mg protein in 60 min at 37°C. MC, myeloid crisis; LC, lymphoid crisis; AP, accelerated phase; CP, chronic phase; C, controls (granulocytes from normal volunteers). The bar indicates the average of the enzyme activities.

Assay of the enzyme activities The cell extract was prepared for poly(A) polymerase assay. The cells were suspended in 20 mM Tris-HC1 buffer (pH 7.0) containing 0.1% Triton X-100 and 2 mM dithiothreitol (DTT), at a concentration of 108 cells/ml, and were frozen and thawed four times. The suspension was centrifuged at 105,000 g using a Hitachi-65P ultracentrifuge (Hitachi Koki Co., Ltd, Japan) for 60 rain at 4°C. The mixture for poly(A) polymerase assay contained 250 mM Tris-HCl (pH 7.0), 2 m M DTT, 2raM MnCI2, i mM 3HATP, 2mg/ml of poly(A) and 100~tg/ml of bovine albumin. The cell extract was added to the mixture and incubated for 30min and 60min at 37°C. The mixture was applied to GF/C filters (Whatman, Ltd, Maidstone, England), washed four times with 5% trichloroacetic acid containing 25 mM sodium pyrophosphate, then washed with ethanol and ether. Finally, filters were counted in a LSC-653 scintillation counter (Aloca Co., Ltd, Tokyo, Japan). Endogenous radioactivity obtained from poly(A)free reaction was subtracted from poly(A)-primed activity. The cells were counted using a Sysmex cell counter, CC110 (Toa Medical Electronics Co., Kobe, Japan). Protein content was assayed, as described previously [13]. The survey of the inhibitors or activators of poly(A) polymerase was done using the mixture of the extracts from three CML cases in the blastic phase and from three cases in the chronic phase. The enzyme activities in each mixture were compared with the calculated values.

Poly(A)-ribonuclease [16], DNA polymerase o~ [12], and TdT [13] activity were assayed, as described previously. In an another study leukemic cells from a CML case in the chronic phase were fractionated with 15 to 31% albumin density gradient, as described previously [12]. Then, the differential count, poly(A) polymerase and D N A polymerase o~activity in each fraction were examined. Effect of TPA or retinoic acid on poly(A) polymerase activity in leukemic cell lines NALM-18 cells [8] (non-T, non-B ALL cell line) were cultured with or without 1.6 × 10 -8 M TPA for 72 h. HL60 cells were cultured with or without 10 6 M 13-cis-retinoic acid for 96 h. The expression of several cell antigens was examined, as described previously [13]. RESULTS T h e addition of 2 m M MnCI2 in the reaction mixture was f o u n d to give maximal p o l y ( A ) p o l y m e r a s e activity when the assay was run at 1-8 m M MgCI 2 or 1-5 m M MnC12 (data not shown). E n z y m e activity was highest at p H 7.0 (range tested, 6.0-8.5) and 250 m M Tris-HC1 (range tested, 25-800 m M ) (data not shown). E n z y m e activity was m a r k e d l y higher with p o l y ( A ) than with oligo A12_18 , transfer R N A ,

Poly(A) polymerase in CML native DNA or activated D N A (DNA treated with pancreatic DNase) (data not shown). The apparent Km of poly(A) and ATP was 0.17 mg/ml and 134 IxM, respectively. The reaction proceeded linearly for 70 min at 37°C. The enzyme activity increased linearly when the cell extracts, at concentrations of 0.30-2.35 mg/ml, were used for the assay. Figure 1 shows poly(A) polymerase activity in CML cases. The mean poly(A) polymerase activity in the leukemic cells from 15 patients with CML in the chronic phase was very low (0.46 --- 0.22 units/5 x 106 cells or 2 . 3 6 - 1 . 1 3 u n i t s / m g protein), similar to that (0.28 - 0.22 units/5 x 106 cells or 2.04 ± 1.33 units/ mg protein) found in the granulocytes of ten normal volunteers. The mean enzyme activity in the leukemic cells from 24 patients with CML in lymphoid or myeloid crisis was significantly higher (t < 0.001), than that in CML patients in the chronic phase. In CML in the blastic phase, poly(A) polymerase activity in myeloid crisis (11.6 -- 4.43 units/5 × 10 6 cells or 74.3 ± 43.7 units/mg protein) was significantly higher (t < 0.001) than that (2.84 ± 1.03 units/ 5 x 106 cells or 26.6 ± 6.51units/mg protein) in lymphoid crisis. Furthermore, leukemic cells from CML patients in the accelerated phase contained moderately high levels of enzyme activity (2.49 -+ 0.55 units/5 x 106 cells or 13.6 ± 1.90 units/ mg protein). Poly(A) polymerase activities were also assayed in AML and ALL cases. The enzyme activities in 14 AML cases (2.37 ± 1.22 units/5 × 10 6 cells or 26.4 ± 10.0 units/mg protein) were significantly higher ( t < 0.001) than those (0.71--0.53 units/ 5 x 106 cells or 8.80 ± 5.46 units/mg protein) in 14 ALL cases. To investigate the presence of inhibitors or activators mixing studies were performed. The levels of the assayed activities did not differ significantly from those of the predicted values (data not shown). In Tables 1 and 2 the levels of poly(A)ribonuclease and DNA polymerase a~ activities are shown along with the level of poly(A) polymerase activity in some of the CML cases. Although a marked difference in poly(A) polymerase activity was observed between CML in the chronic phase and in the blastic or accelerated phase, the level of DNA polymerase o: activity was comparable between two groups. In addition, the level of poly(A)-ribonuclease activity in CML patients in the blastic phase was slightly higher ( t < 0.05) than that in CML patients in the chronic phase. In a CML case in the chronic phase, leukemic cells were fractionated with an albumin density gradient. As shown in Table 3, poly(A) polymerase activities in fractions rich in immature myeloid cells were higher than those in fractions rich in mature myeloid cells. In addition, the increasing level of poly(A) polymerase activity in

275

AML cases (n = 14, r = 0.83) and CML cases (n = 17, r = 0.80) in the myeloid crisis or the accelerated phase roughly corresponded to the increasing percentage of the blasts. However, as shown in Tables 2 and 3, the level of poly(A) polymerase activity in leukemic cells did not necessarily correlate with the level of DNA polymerase tr activity. Table 4 shows the effect of TPA on poly(A) polymerase activity and the expression of several cell antigens in a lymphoid leukemia cell line (NALM-18). Poly(A) polymerase activity in TPA-treated leukemia cells was decreased significantly, along with the decrease in the ratio of J5-, B4- and TdT-positive cells and the increase in B~-positive cells. Similarly, the significant decrease in poly(A) polymerase activity was observed in HL60 cells which was induced to differentiate by the exposure to 10-6M retinoic acid for 96 h (data not shown). DISCUSSION Poly(A) metabolism enzymes are involved in the turnover of poly(A) sequences in eukaryotic mRNA. Thus, a qualitative or quantitative change in the poly(A) metabolism enzymes may indirectly modulate the protein synthesis by affecting the poly(A) sequences in mRNA. In this study, we examined the optimal conditions for poly(A) polymerase assay using HL-60 cell line, then, assayed the enzyme activity in various leukemia cases. Trangas et al. [16] reported that poly(A) polymerase activity in acute leukemia was statistically higher than that in chronic lymphocytic leukemia (CLL) and that poly(A) polymerase activities in CLL cases were related to the clinical stage or the pattern of bone marrow involvement [10]. However, there are still no reports concerning poly(A) polymerase activities in CML cases. In this study, poly(A) polymerase activity in CML in the chronic phase was low, similar to that in granulocytes. However, the enzyme activity was moderately high in CML in the accelerated phase and markedly elevated in the blastic phase. The results of the mixing experiments suggested that there were no inhibitors or activators for this enzyme in leukemic cells from CML cases. The poly(A) sequence of mRNA in the eukaryotic cells seems to be controlled by poly(A)-anabolic and catabolic enzymes [9, 14]. Therefore, a change in poly(A)-ribonuclease activity may be responsible for the increased 3H-AMP incorporation in cases of CML in the blastic phase. However, the assay of poly(A)-ribonuclease activity in CML cases eliminates this possibility. In addition, the enzyme activity was significantly higher in cases of CML in the myeloid crisis than in

276

RYUHEI SASAKIel al. TABLE 1. POLY(A) RIBONUCLEASE AND POLY(A) POLYMERASE ACTIVITIES IN C M L CASES

CML (Chronic phase) Patient T.S. Y.K. K.O. R.N. Y.Y. I.N. S.S. S.K.

CML (Blastic phase)

Poly(A) ribonuclease (units/3 x 106)

Poly(A) polymerase (units/5 x 106)

0.61 1.17 1.04 0.82 0.76 1.28 1.11 1.19 1.00 ± 0.24*

0.29 0.72 0.48 0.84 I).22 /).43 0.66 0.30 0.49 ± 0.23t

Patient Y.S. K.F. F.M. Y.W. M.K. C.S. T.I. K.F.

Poly(A) ribonuclease (units/3 x 106)

Poly(A) polymerase (units/5 x 106)

1.40 1.05 1.08 1.14 1.43 1.36 1.48 1.20 1.27 -+ 0.17"

18.64 2.94 4.85 11.83 6.62 20.27 4.88 2.94 9.12 + 6.53t

* Mean + standard deviation (n = 8). A significant difference (p < 0.05) exists between CML in the chronic phase and CML in the blastic phase. t A significant difference (p < 0.01) exists between CML in the chronic phase and CML in the blastic phase.

TABLE 2. D N A POLYMERASE Ol AND POLY(A) POLYMERASE ACTIVITIES IN C M L CASES

CML (Blastic phase or accelerated phase)

CML (Chronic phase) Patient S.K. Y.Y. R.N. I.A. S.H. Y.K. I.N. G.O.

D N A polymerase o: (units/10 s)

Poly(A) polymerase (units/5 x 106)

0.20 0.20 0.03 0.24 0.25 0.13 0.04 0.05 0.14 -+ 0.09t

0.30 0.22 0.84 0.61 0.70 0.72 0.43 0.48 0.54 ± 0.225

Patient K.W. T.O. F.M.* T.H. Y.W.* S.S. K.F.* T.I.*

D N A polymerase ol (units/108)

Poly(A) polymerase (units/5 × 10 6)

0.05 0.12 0.03 0.09 0.27 0.22 0.12 0.17 0.13 -+ 0.08t

2.49 1.71 4.85 2.25 11.83 3.11 2.94 4.88 4.26 ± 3.272

* These patients had CML in the blastic phase, while others had CML in the accelerated phase. t Mean +- standard deviation (n = 8). No significant difference was found between CML in the chronic phase and CML in the blastic or accelerated phase. $ A significant difference (p < 0.01) exists between CML in the chronic phase and CML in the blastic or accelerated phase.

TABLE 3. POLY(A) POLYMERASE AND D N A POLYMERASE 0¢ACTIVITY IN EACH FRACTION OF THE BLOOD FROM A C M L CASE

Differential count (%) Fraction

Mbl

proM

M

MM

Band + Seg

Others

Poly(A) polymerase (units/5 x 106)

D N A polymerase o~ (units/108)

1. 2. 3. 4. 5.

0 0 0.5 10.6 16.8

1.5 2.7 9.7 15.6 11.4

22.1 39.9 78.5 64.7 49.1

38.5 40.4 5.2 4.t 14.1

37.4 15.4 5. l 2.4 5.9

0.5 1.6 1.0 2.6 2.7

0.60 0.68 1.53 2.22 3.34

0.03 0.05 0.12 0.13 0.12

Leukemic cells from a CML case in the chronic phase were fractionated with 15-31% albumin density gradient. The differential count, poly(A) polymerase and D N A polymerase ~xactivity in each fraction were examined. Mbl, myeloblast; proM, promyelocyte; M, myelocyte; MM, metamyelocyte; Band, banded neutrophils; Seg, segmented neutrophils.

Poly(A) polymerase in CML

277

TABLE 4. EFFECT OF T P A ON POLY(A) POLYMERASE ACTIVITY AND THE EXPRESSION OF SEVERAL CELL ANTIGENS IN N A L M - 1 8 CELLS

Poly(A) polymerase TdT

I2 J5 TdT BI B4

-TPA

+TPA

0.86 units/5 x 106 cells 174 units/10 s cells

0.23 units/5 x 106 cells 21.8 units/10 s cells

- T P A (% positive cells)

+TPA (% positive cells)

94.0% 58.9% 66.0% 7.52% 42.3%

89.1% 27.6% 36.9% 22.9% 24.9%

NALM-18 cells were cultured with or without 1.6 × 10-8M TPA for 72 h at 37°C. Poly(A) polymerase activity and the expression of several cell antigens were examined. A panel of monoclonal antibodies was obtained from Coulter Immunol., Hialeah, FL. The immunoadsorbent purified rabbit anti-TdT sera were used for the analysis of TdT-positive cells. the lymphoid crisis. Also, the enzyme activity in A M L cases was significantly higher than that in A L L cases. This seems to be consistent with the difference in poly(A) polymerase activities between CML in the myeloid crisis and in the lymphoid crisis. Miiller et al. [9] reported that poly(A) polymerase activity in synchronized mouse L-cells remained essentially constant, independent of cell cycle, while the poly(A) catabolic enzyme activities increased parallel with D N A synthesis. On the contrary, Adolf and Swetly [1] reported that poly(A) polymerase activity in erythroleukemic mouse spleen cells was low in resting cells and increased in the S-phase. In our study, the level of D N A polymerease oLactivity in CML in the chronic phase was comparable with that in CML in the accelerated or the blastic phase. In leukemic cells fractionated with albumin density gradient poly(A) polymerase activity in fractions rich in immature myeloid cells was higher than that in fractions rich in mature myeloid cells. In these fractions the level of poly(A) polymerase activity did not necessarily correlate with the level of D N A polymerase oi activity. In addition, the level of poly(A) polymerase activities was correlated roughly with the percentage of the blasts in many leukemia cases. Also, T P A and retinoic acid are well known as the inducers for the differentiation of various leukemic cells. In the TPA-treated lymphoid leukemia cell line poly(A) polymerase activities were decreased along with the altered expression of several cell markers suggesting the differentiation of the lymphoid leukemia cells. Similar change in poly(A) polymerase activity was observed in HL-60 cells exposed to retinoic acid. Therefore, these results show that the

poly(A) polymerase activity changes, depending on the maturation of leukemic cells, though it remains to be elucidated whether the change in this enzyme activity is causally related to the differentiation processes of leukemic cells. In addition, the significant difference in the poly(A) polymerase activities observed in various phases of CML cases shows that the assay of this enzyme activity may be useful for the early detection of the exacerbation of CML cases.

Acknowledgements--The technical assistance of Miss T. Kumakura is gratefully acknowledged. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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metabolizing enzyme levels during induction of differentiation in a human leukemia T-cell line with phorbol ester. J. natn. Cancer Inst. 78, 407. 12. Sasaki R. & Sakamoto S. (1977) The function and clinical significance of DNA polymerases in human hematopoietic cells. Acta Haemat. jap. 40, 1104. 13. Sasaki R., Takaku F., Lin Y-L. & Bollum F. J. (1982) Sialyltransferase activity in leukemia. Leukemia Res. 6, 197. 14. Schr6der H. C., Zahn R. K., Dose K. & Miiller W. E. G. (1980) Purification and characterization of a poly(A)-specific exoribonuclease from calf thymus. J. biol. Chem. 255, 4535. 15. Simantov R. & Sachs L. (1975) Induction of polyadenylate polymerase and differentiation in neuroblastoma cells. Eur. J. Biochem. 55, 9. 16. Trangas T., Courtis N., Pangalis G. A., Cosmides H. V., Ioannides C., Papamichail M. & Tsiaparis C. M. (1984) Polyadenylic acid polymerase activity in normal and leukemic human leukocytes. Cancer Res. 44~ 3961.