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Deoxycytidylate deaminase activity and its template stability in human acute lymphoblastic and myeloblastic leukaemic cells
Europ. d. Cancer Vol. 12, pp. 611-616. Pergamon Press 1976. Printed in Great Britain
Deoxycytidylate Deaminase Activity and its Template Stability in Human Acute Lymphoblastic and Myeloblastic Leukaemic Cells* MARIA GRAZIA BERNENGO and LUIGI PEGORARO Sezione di Ematologia, Clinica Medica Generale dell' Universita di Torino, Torino, Italia Abstract--The activity of deoxycytidylate (dCMP) deaminase, an enzyme of the main pathway of the D N A synthesis and the effects of actinomycin D upon its template stability were studied in the peripheral blast cells of 11 acute leukaemia patients. The relationship between e:~zyme activity, template stabzhty, cytochemical characteristics and proliferative activity ~f the blast cells was also investigated. By employing current cytochemical techniques, 6 of the 11 cases were classified as lymphoblastic and 5 as myeloblastic. The proliferative activity was evaluated with autoradiographic technique following short incubation with SH-thymidine. Acute myeloblastic leukaemia (AML) cells displayed higher enzyme activity than acute lymphoblastic leukaemia (ALL) cells. In addition, a direct correlation between the enzyme activity and the proliferative activity was shown in A M L cells. dCMP deaminase l!emplate stability was assayed at the end of a 9 hr culture time in presence of actinomycin D at a dose which inhibited 90 ~o of 3H-uridine incorporation into the RNA of both A M L and ALL cells. While none of the cases of A M L showed significant change of the dCMP deaminase activity after actinomycin D treatment, in all cases Of ALL a marked decrease of the enzyme activity was observed, suggesting that, at least in the steady staJ:econditions, the A M L and the ALL cells have a different rate of degradation of the RNA templates specificfor the dCMP deaminase and that the rate of degradation is related to the proliferative activity of the cells. In fact, in the ALL cases a significant correlation was observed between the proliferative activity and the decrease of the enzyme activity induced by actinomycin D.
INTRODUCTION
pathway have been extensively investigated in the acute leukaemias and thoroughly reviewed [6]. The main aim of these studies was the search for either differences between normal leukocytes and leukaemic cells or for the changes induced by chemotherapic agents in the activity of these enzymes in leukaemic cells. O n the other hand, since the treatments for ALL and for A M L are becoming increasingly specific, the finding of differences between these two types of leukaemia might lead to a more rational design of therapeutic schemes. d C M P deaminase is an enzyme of the main pathway leading to the synthesis of thymidylate. In the present communication we report on studies on the activity of this enzyme in acute leukaemia cells and on changes of this activity after actinomycin D treatment. Acute leukaemia cases were classified, according to
THE CLASSIFICATION of the myeloblastic and lymphoblastic forms of human acute leukaemias is mainly based upon morphological and cytochemical criteria. In addition, the blast cells of these forms have been shown to exhibit different cytogenetic [1, 2] and kinetic [3, 4] characteristics. However, with few exceptions [5], biochemical studies on human acute leukaemias have been done without taking into separate consideration the lymphoblastic and the myeloblastic forms. Because of their relationship to chemotherapy the enzymes of the DNA synthetic
Accepted 20 February 1976. *Supported by Grant 71.00823.04 from CNR Rome. 6/1
612
Maria Grazia Bernengo and Luigi Pegoraro
the current morphological and cytochemical criteria, as ALL or AML. In each case the dCMP deaminase activity was compared both with cytochemical characteristics and with the proliferative activity of the blast cells. MATERIAL AND METHODS
The cells studied were obtained from the peripheral blood of 11 acute leukaemia untreated patients. The age of the patients ranged between 4 and 75 y. Peripheral leukocyte levels ranged from 30,000 to 274,000 of which more than 67% were blast cells. The heparinized venous blood was allowed to sediment at 37°C and the leukocyte layer was separated, diluted with M E M (Gibco) containing 20% of autochtonous plasma to obtain a cell concentration of 3-6 x 106 cells/ml, and distributed in 7 ml aliquots into glass screw cap flasks of 25 ml total capacity. The cultures were incubated at 37°C for 9 hr in atmosphere of 95% balanced air and 5% CO2. Actinomycin D (a gift from Merck, Sharp & Dohme), when used, was added to a final concentration of 0.5 pg/ml. All cultures were done in triplicate. The viability of the cells when the cultures were arrested was checked by their ability to exclude the dye trypan blue [7]. The cultures were harvested by centrifugation and contaminating erythrocytes lysed by osmotic shock [8]. This treatment did not produce any significant change in the enzyme activity. For the determination of deoxycytidylate deaminase activity [9] approximately 4 x 107 ceils were homogenized in 0.5 ml of 0.15 M KC1, 3 m M 2-mercaptoethanol, 10 m M TrisHC1 pH8 at 20°C, 0.31 m M dCTP, with a motor driven glass homogenizer of 1 ml total capacity. The homogenate was centrifuged at 30,000 g at 4°C for 15 min in a Sorvall refrigerated ultracentrifuge and the supernatant used as the source of the enzyme. The reaction contained, in a final volume of 0.25 ml: dCTP (Sigma) 20 nmoles, MgC12 .5 pmoles, 2mercaptoethanol 0.75 #moles, NaF 3-7/~moles, Tris-HC1 (pH 8 at 20°C) 21 pmoles, 3Hd C M P (Amersham) 25/~moles (2"77 x 106 dis/ rain) and enzyme extract consisting of 20-80 #g of protein. After 10 rain of incubation at 37°C the reaction was stopped by addition of 0.25 ml of 0.3 N perchloric acid. The enzyme activity was linear up to 60 rain and was proportional to a broad range of protein concentration. d C M P and d U M P were separated by descending chromatography on DEAE 81 Whatman paper strips with 4 N formic acid and 0.1 N ammonium formate as the solvent.
The strips were cut into 2 cm sections and counted in omnifluortoluene (NEN). The protein concentration of the enzyme extract was determined by the method of Lowry et al. [10]. In order to study RNA synthesis cultures were incubated for 9 hr with and without actinomycin D. Uridine aH-5-T (24.6 Ci/mM) was added 2 hr before harvesting the cultures at a concentration of 10 #Ci/ml. The cells were harvested and washed twice with cold buffered saline solution. The pellet was then suspended in 5% cold TCA. After 30 rain at 0°C the precipitate was filtered through a millipore filter and the filters washed 3 times with 5% TCA. The filters were dissolved in 1 ml cellosolve and mixed with 15 ml of liquifluor for the determination of the radioactivity in a Nuclear Chicago scintillation spectrometer model Mark 1. For the determination of the labelling indices 1 ml aliquot, of peripheral blood were incubated for 1 hr with 3H-thymidine (Amersham, 6.7 Ci/mM) at the concentration of 1 ~Ci/M1. Slides were processed for autoradiography [11] and the fraction of cells in S determined on at least 1000 cells. The peroxidase reaction was determined by the method of Hayhoe [12], the Sudan black reaction by the method of Sheehan and Storey [13], and the periodid acid Schiff(PAS) reaction by the method of Hayhoe et al. [12]. Positive peroxidase reactions were considered those where weak diffuse granules or a tinge confined to Auer rods were present in the blast cells; positive Sudan black reactions those where blast cytoplasm showed variable amounts of sudanophilic granules or else only Sudan positive Auer rods; positive PAS reactions those where there was a crown of cytoplasmic granules or a single block of glycogen even if this particular type of staining reaction was present in only a very small number of immature cells. On the contrary, if blast ceils were completely negative or if they showed a weak diffuse tinge or, more exceptionally, if their cytoplasm contained PAS positive Auer rods, PAS reactions were considered negative [12]. RESULTS
The main features of the cases used for the present study are listed in Table 1. Leukaemias were classified as lymphoblastic or myeloblastic according to the following criteria of recognition. The group of lymphoblastic leukaemias included those with a high nucleocytoplasmic ratio, absence of Auer rods and of
Deoxycytidylate Deaminase Activity
613
Table 1. Clinical and cytochemieal data of the acute leukaemia cases
Case No. Acute myeloblastic ]eukaemia
Acute lymphoblastie leukaemia
Sex
WBC/ cumm x 10 a
% of blasts
Sudan black
Peroxidase
PA S
6 22 I0 70 70
F F M F F
66 274 14 184.6 15
90 87 84 86 67
+ + + + +
+ + + + +
------
75 6 4 45 65 4
F F M M F F
51 40 16 16 171 235
85 97 86 93 84 93
-------
-------
+ + + + + +
Patient
Age . (years)
1 2 3 4 5
A.P. C.V. S.E. R.A. C.M.
6
P.F. A.V. C.P. R.M. R.A. M.O.
7 8 9 10 11
other intracellular structures, negative peroxidase and Sudan black reactions and positive PAS reaction. T h e myeloblastic forms were considered those with a lower nucleo-cytop l a s m i c r a t i o , p r e s e n c e o f A u e r rods a n d / o r o f azurofil granulations, positivity of peroxidases and Sudan black reactions and negativity of PAS reaction. T h e results o f t h e d e t e r m i n a t i o n o f d C M P d e a m i n a s e a c t i v i t y a n d o f t h e effect o f a c t i n o m y c i n D u p o n the e n z y m e activity are shown i n T a b l e 2. T h e v a l u e s o f t h e a v e r a g e l a b e l l i n g i n d e x o f t h e b l a s t cells p o p u l a t i o n o f e a c h case a r e also r e p o r t e d . T h e effect o f a c t i n o m y c i n D is s h o w n i n t h e t a b l e as t h e p e r c e n t a g e o f i n h i b i t i o n o f e n z y m e a c t i v i t y , t a k i n g as 100 t h e enzyme activity of the untreated cultures. The effectiveness o f t h e dose e m p l o y e d w a s c h e c k e d
Table 2.
by its ability to reduce uridine incorporation into RNA. In both lymphoblastic and myeloblastic leukaemias actinomycin D inhibited uridine incorporation more than 90%. Furthermore, under these conditions, actinomycin D did not produce any gross morphological change in the cells nor differences in the cell viability, as judged by the trypan blue exclusion test, between treated and control cultures. Table 2 shows that the dCMP deaminase activity is significantly (P <0-05) higher in AML than in ALL cells. In addition, a close correlation was observed between dCMP deaminase activity and the percentage of labelled cells in the AML forms (Fig. 1). Enzyme activity was directly proportional to the proliferative activity. On the contrary, in acute
Deoxycytidylate deaminase activity and inhibition of the enzyme activity following actinomycin D treatment *mgmoles of dCMP deaminated/min/ % of 1"Labelling Case mg of protein inhibition indices
Acute myeloblasdc leukaemia
Acute lymphoblastic leukaemia
1
13.0+
2 3 4 5
20.8 + 0.7 27.4+ 0.1 35.9 + 19-4 48.6+ 7.1
6 7 8 9 10 11
24 + 23.6 + 4.8 + 2.5 + 12.4+ 9.4+
1.
1.3 3.5 2.5 1.1 0.3 0.3
0 0 0 4
2.6 3.1 3.7 4.3 5
73 68.5 68 42 14 10-3
1 2.4 2"2 9-8 18.4 19.6
0
*Each number is the average of three determinations. ~Percentage of 3 H - T d R labelled blast cells.
Maria Grazia Bernengo and Luigi Pegoraro
614
lymphoblastic leukaemia d C M P deaminase activity exhibited a greater range of variability and was not related with the cell proliferative rate. The effect of actinomycin D upon the activity of the enzyme revealed different behaviour pattern between the two types of leukaemias, d C M P deaminase activity in all cases of A M L was not affected by the 9-hr actinomycin D treatment, while in all cases of ALL a decrease of enzyme activity was ob-
$
~ ..4 z_.
-t m
2
,J
0
m
I
I
20
I
I
40
I
13"11.1 MOLES dCMP DEAMINATED//MIN/MG
I
60
OF PROTEIN
Fig. 1. Correlation between the percentage of labelled blast cells (ordinate) and the dCMP deaminase activity (abscissa), expressed in mltmoles deaminated/minute[mg of protein, in the A M L cases.
I
I 3
% OF INHIBITION
Fig. 2. Correlation between the percentage of labelled blast cells (ordinate) and the percentage of inhibition of the dCMP deaminase activity by actinomycin D (abscissa) in the ALL
cases. (u - - 0"15%).
served. Since the degree of inhibition varied from case to case a possible relationship of the actinomycin effects with the proliferative activity of the ALL cells was investigated. A close correlation (Fig. 2) was observed between the template stability and the labelling indices.
DISCUSSION The results of this study show that the blast cells o f A M L and of ALL possess a different ability to deaminate dCMP. In fact, the activity of d C M P deaminase was found to be higher in the myeloblastic than in the lymphoblastic forms in spite of the fact that the latter possess a higher proportion of cells in the S phase of the cell cycle. In A M L a direct correlation was found between the proliferative activity and the enzyme activity of the blast cells. Increased activity of some of the enzymes of the DNA synthetic pathway, including d C M P deaminase have been shown to occur preceding or concomitantly with DNA synthesis in a number of systems [14, 15]. It is not surprising therefore that those A M L cases with a higher fraction of cells in S and hence, with a higher demand of T M P synthesis, also possess a higher d C M P deaminase activity. It is not clear, however, why in the ALL forms such correlation is absent. It is possible that this represents an imbalance in the regulation of the enzyme molecules synthesis or activity. However, one must also consider the possibility that the group of ALL is heterogeneous in the sense that m a n y subgroups of ALL are included in it. Cytochemical investigations seems to support this view [16]. The main difference found in the present study between the A M L and ALL forms concerns the stability of the enzyme activity to actinomycin D treatment. Actinomycin D, at the dose we used, almost completely inhibits R N A synthesis, in both A M L and ALL cells, and hence prevents the synthesis of new templates. It is therefore likely that a decrease of d C M P deaminase activity in the treated cultures is due to depletion of the RNA templates specific for this enzyme. The fact that after 9 hr of actinomycin D treatment the activity of d C M P deaminase in A M L cells it is unchanged while in ALL cells it is strongly decreased suggests that the former possess a more stable template for this enzyme. The stability of the templates of the enzymes of the DNA synthetic pathway has been extensively studied, with the aid of actinomycin D, both in systems of stimulated DNA synthesis and in continuously
Deoxycytidylate Deaminase Activity
dividing normal and neoplastic cells. In quiescent or Go cells RNA templates were found, in most instances, to be more stable than in cells stimulated to undergo to DNA synthesis [1721]. We have shown that the treatment of P H A stimulated normal lymphocytes with actinomycin D for 9 hr preceding the maximum DNA synthesis decreases to 50% of the d C M P deaminase activity. Conversely, in unstimulated lymphocytes, treatment for 9 hr with actinomycin D does not affect the enzyme activity [22]. The stability o f d C M P deaminase activity to actinomycin D treatment has also been found to be greater in normal than in regenerating liver [17]. In rapidly proliferating normal and neoplastic cells little or no effect of actinomycin D has been shown upon the activity of the enzymes of the DNA synthetic pathway [23-25]. In the present study A M L cells, when treated with actinomycin D, behave in the same way as Novikoff hepatoma and Ehrlich ascites cells, while, in most cases, ALL cells are very similar to cells stimulated to DNA synthesis. Furthermore, in the A L L a correlation between the d C M P deaminase template lability and the proliferative activity of the blast cells was found, suggesting that the half fife of the
R N A templates is directly proportional to the percent of cycling cells. Since the enzyme level within a cell is dependent not only upon the stability of its templates but also upon the turnover rate of the enzyme protein itself, the difference between A M L and ALL ceils could depend upon a different half life of the d C M P deaminase molecules. The fact that other systems such as normal adult and regenerating fiver possess equally stable thymidine kinase but different turnover for the template of this enzyme may suggest that also A M L and ALL cells have a similar pattern [26]. Unfortunately, our attempts to evaluate the half life of d C M P deaminase with cycloeximide treatment in acute leukaemic cells were unsuccessful. We cannot therefore assert from our results whether the difference between the ALL and the A M L cells lies at the level of transcription or at the level of degradation of the enzyme molecules. Our data concerning the d C M P deaminase have shown differences between A M L and ALL both in the activity of the enzyme and in effects of actinomycin D upon this activity. It may be worth further studies to see whether this difference is limited to d C M P deaminase or is it common to other enzymes of the DNA synthetic process.
REFERENCES
1. 2. 3. 4.
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615
A.A. SANDBERG,T. ISHIHARA,Y. K.IKUCHIand L. H. CROSSWmTE, Cromosomal differences among the acute leukemias. Ann. N.Y. Acad. Sci. 113, 663, (1964). 1.. E. RXISMAN,W. W. ZUELZERand R. I. THOMPSON, Further observation on the role of aneuploidy in acute leukemia. Cancer Res. 24, 1448 (1964). F. GAVOSTO, A. PILERI, A. PONZONE, P. MASERA, R. P. TAROCCO and V. GAUUTTI, Different blast kinetics in acute myeloblastic and lymphoblastic leukaemia. Acta. Haemat. 41, 215 (1969). F. GAVOSTO,Observations on the kinetics of acute lymphoblastic leukaemia in relation to its division into subvarietes. In Recent Results in Cancer Research. (]Edited by G. MATHE,P. POUILLARTand L. SCI-IWARZEI~.RO)Vol. 43, p. 76 (1972). I). ROB~.RTSand T. C. HALL,Enzyme activities and deoxynucleoside utilization of leukemic leukocytes in relation to drug therapy and resistance. Cancer Res. 29, 166 (1969). R . C . GALLO,Synthesis and metabolism of DNA and DNA precursors by human normal and leukemic leukocytes. Acta Haemat. 45, 136 (1971). A. 1~. PAPPENHEIMER,Experimental studies upon lymphocytes. I. The reactions of lymphocytes under various experimental conditions. J. exp. Med. 25, 633 (1916). Y.R.~INOWITZ, DNA polymerase and carbohydrate metabolizing enzyme content of normal and leukemic glass column separated leukocytes. Blood 27, 779 (1962). F MALEY and G. F. MALEY, Nucleotide interconversions. II. Elevation of deoxycytidylate deaminase and thymidylate synthetase in regenerating rat liver, or. biol. Chem. 235, 2968 (1960). O . H . LowRY, N. J. ROSEBROUOH,A. L. FAgR and R. J. RANDALL, Protein measurement with the Folin phenol reagent. J. bwl. Chem. 193, 265 (1951).
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F. GAVOSTO,A. PILERI, C. BACHIand L. PEOORARO,Proliferation and maturation defect in acute leukaemia cells. Nature (Lond.) 203, 92 (1964). F . G . J . HAYHOE,D. Q UAOLINOand R. DOLL, The cytology and cytochemistry of acute leukaemias. M. R. C. Specml Report Sines n. 304, p. 16, London (1964). H . L . SHEEHANand G. W. STOREY, An improved method of staining leukocyte granules with Sudan black. J. Path. Bact. 59, 336 (1947). A.S. GELBARD,J. H. KIM and A. G. PEREZ, Fluctuations in deoxycytidine monophosphate deaminase activity during the cell cycle in synchronous populations of HeLa cells. Bzoch~m. biophys. Acta 182, 564 (1969). E. STUBBLEFIELDand S. MURPHREE, Synchronized mammalian cell cultures. II. Thymidine kinase activity in Colcemid synchronized fibroblasts. Exp. Cell Res. 48, 652 (1967). G. MATHE, P. POUILLART, M. STERESCU,J. L. AMIEL, L. SC~WARZENBERG, M. SCHNEIDER,M. HAYAT,F. DE VASSAL,C. JASMINand M. LAFLEUR, Subdivision of classical varieties of acute leukemia. Correlation with prognosis and cure expectancy. Rev.franc. Etud. din. biol. 16, 554 (1971) I. LIEBERMAN,R. ABRAMSand P. OVE, Changes in the metabolism of ribonucleic acid preceding the synthesis of deoxyribonucleic acid in Mammalian cells cultured from the animal, or. biol. Chem. 238, 2141 (1963). R . L . HOLTZER,A. ODA and M. CHIGA,Effect of actinomycin D on deoxycytidylate deaminase activity of rat liver after partial hepatectomy. Lab. Invest. 13, 1514 (1964). H . C . PITOT, C. PERmNO, LAMARJR. and A. L. KENNAN, Template stability of some enzymes in rat liver and hepatoma. Proc. nat. Acad. Sei. (Wash.) 51~ alo (1964). G.F. MALEY, M. G. LORENSONand F. MALEY, Inhibitors of protein synthesis: effect on the levels of deoxycytidylate deaminase, thymidylate synthetase, and thymidine kinase in regenerating rat liver. Biochem. biophys. Res. Comm. 18, 364 (1965). L. PEOORARO and R. BASEROA, Time of appearance of deoxythymidylate kinase and deoxythymidylate synthetase and of their templates in isoproterenolstimulated deoxyribonucleic acid synthesis. Lab. Invest. 229 266 (1970). L. PEOORAROand M. G. BERNENOO,Thymidine kinase, deoxycytidine kinase and deoxycytidylate deaminase activities in phytohaemagglutinin stimulated human lymphocytes. Exp. Cell Res. 68~ 283 (1971). J . S . ROTH, Increased stability of templates for some enzymes of DNA synthesis in tumors: Deoxycytidylate deaminase, thymidine and thymidine kinase. Life Sci. 3, 1145 (1964). R. BASEROA,R. D. ESTENSEN,R. O. PETERSEN and J. P. LAYDE,Inhibition of DNA synthesis in Ehrlich ascites cells by Actinomycin D. I. Delayed inhibition by low doses. Proc. nat. Acad. Sci. (Wash.) 54, 745 (1956). E. BRESNICK,S. S. WILLIAMSand H. MOSSE, Rates of turnover of deoxythymidine kinase and of its template RNA regenerating and control liver. Cancer Res. 27, 469 (1967). E. Bm~SNICKand S. S. BURLESON,Rates of turnover of deoxythymidine kinase and of its template RNA in Novikoff Hepatoma and neonatal liver. Cancer Res. 30~ 1060 (1970).
Deoxycytidylate Deaminase Activity and its Template Stability in Human Acute Lymphoblastic and Myeloblastic Leukaemic Cells* MARIA GRAZIA BERNENGO and LUIGI PEGORARO Sezione di Ematologia, Clinica Medica Generale dell' Universita di Torino, Torino, Italia Abstract--The activity of deoxycytidylate (dCMP) deaminase, an enzyme of the main pathway of the D N A synthesis and the effects of actinomycin D upon its template stability were studied in the peripheral blast cells of 11 acute leukaemia patients. The relationship between e:~zyme activity, template stabzhty, cytochemical characteristics and proliferative activity ~f the blast cells was also investigated. By employing current cytochemical techniques, 6 of the 11 cases were classified as lymphoblastic and 5 as myeloblastic. The proliferative activity was evaluated with autoradiographic technique following short incubation with SH-thymidine. Acute myeloblastic leukaemia (AML) cells displayed higher enzyme activity than acute lymphoblastic leukaemia (ALL) cells. In addition, a direct correlation between the enzyme activity and the proliferative activity was shown in A M L cells. dCMP deaminase l!emplate stability was assayed at the end of a 9 hr culture time in presence of actinomycin D at a dose which inhibited 90 ~o of 3H-uridine incorporation into the RNA of both A M L and ALL cells. While none of the cases of A M L showed significant change of the dCMP deaminase activity after actinomycin D treatment, in all cases Of ALL a marked decrease of the enzyme activity was observed, suggesting that, at least in the steady staJ:econditions, the A M L and the ALL cells have a different rate of degradation of the RNA templates specificfor the dCMP deaminase and that the rate of degradation is related to the proliferative activity of the cells. In fact, in the ALL cases a significant correlation was observed between the proliferative activity and the decrease of the enzyme activity induced by actinomycin D.
INTRODUCTION
pathway have been extensively investigated in the acute leukaemias and thoroughly reviewed [6]. The main aim of these studies was the search for either differences between normal leukocytes and leukaemic cells or for the changes induced by chemotherapic agents in the activity of these enzymes in leukaemic cells. O n the other hand, since the treatments for ALL and for A M L are becoming increasingly specific, the finding of differences between these two types of leukaemia might lead to a more rational design of therapeutic schemes. d C M P deaminase is an enzyme of the main pathway leading to the synthesis of thymidylate. In the present communication we report on studies on the activity of this enzyme in acute leukaemia cells and on changes of this activity after actinomycin D treatment. Acute leukaemia cases were classified, according to
THE CLASSIFICATION of the myeloblastic and lymphoblastic forms of human acute leukaemias is mainly based upon morphological and cytochemical criteria. In addition, the blast cells of these forms have been shown to exhibit different cytogenetic [1, 2] and kinetic [3, 4] characteristics. However, with few exceptions [5], biochemical studies on human acute leukaemias have been done without taking into separate consideration the lymphoblastic and the myeloblastic forms. Because of their relationship to chemotherapy the enzymes of the DNA synthetic
Accepted 20 February 1976. *Supported by Grant 71.00823.04 from CNR Rome. 6/1
612
Maria Grazia Bernengo and Luigi Pegoraro
the current morphological and cytochemical criteria, as ALL or AML. In each case the dCMP deaminase activity was compared both with cytochemical characteristics and with the proliferative activity of the blast cells. MATERIAL AND METHODS
The cells studied were obtained from the peripheral blood of 11 acute leukaemia untreated patients. The age of the patients ranged between 4 and 75 y. Peripheral leukocyte levels ranged from 30,000 to 274,000 of which more than 67% were blast cells. The heparinized venous blood was allowed to sediment at 37°C and the leukocyte layer was separated, diluted with M E M (Gibco) containing 20% of autochtonous plasma to obtain a cell concentration of 3-6 x 106 cells/ml, and distributed in 7 ml aliquots into glass screw cap flasks of 25 ml total capacity. The cultures were incubated at 37°C for 9 hr in atmosphere of 95% balanced air and 5% CO2. Actinomycin D (a gift from Merck, Sharp & Dohme), when used, was added to a final concentration of 0.5 pg/ml. All cultures were done in triplicate. The viability of the cells when the cultures were arrested was checked by their ability to exclude the dye trypan blue [7]. The cultures were harvested by centrifugation and contaminating erythrocytes lysed by osmotic shock [8]. This treatment did not produce any significant change in the enzyme activity. For the determination of deoxycytidylate deaminase activity [9] approximately 4 x 107 ceils were homogenized in 0.5 ml of 0.15 M KC1, 3 m M 2-mercaptoethanol, 10 m M TrisHC1 pH8 at 20°C, 0.31 m M dCTP, with a motor driven glass homogenizer of 1 ml total capacity. The homogenate was centrifuged at 30,000 g at 4°C for 15 min in a Sorvall refrigerated ultracentrifuge and the supernatant used as the source of the enzyme. The reaction contained, in a final volume of 0.25 ml: dCTP (Sigma) 20 nmoles, MgC12 .5 pmoles, 2mercaptoethanol 0.75 #moles, NaF 3-7/~moles, Tris-HC1 (pH 8 at 20°C) 21 pmoles, 3Hd C M P (Amersham) 25/~moles (2"77 x 106 dis/ rain) and enzyme extract consisting of 20-80 #g of protein. After 10 rain of incubation at 37°C the reaction was stopped by addition of 0.25 ml of 0.3 N perchloric acid. The enzyme activity was linear up to 60 rain and was proportional to a broad range of protein concentration. d C M P and d U M P were separated by descending chromatography on DEAE 81 Whatman paper strips with 4 N formic acid and 0.1 N ammonium formate as the solvent.
The strips were cut into 2 cm sections and counted in omnifluortoluene (NEN). The protein concentration of the enzyme extract was determined by the method of Lowry et al. [10]. In order to study RNA synthesis cultures were incubated for 9 hr with and without actinomycin D. Uridine aH-5-T (24.6 Ci/mM) was added 2 hr before harvesting the cultures at a concentration of 10 #Ci/ml. The cells were harvested and washed twice with cold buffered saline solution. The pellet was then suspended in 5% cold TCA. After 30 rain at 0°C the precipitate was filtered through a millipore filter and the filters washed 3 times with 5% TCA. The filters were dissolved in 1 ml cellosolve and mixed with 15 ml of liquifluor for the determination of the radioactivity in a Nuclear Chicago scintillation spectrometer model Mark 1. For the determination of the labelling indices 1 ml aliquot, of peripheral blood were incubated for 1 hr with 3H-thymidine (Amersham, 6.7 Ci/mM) at the concentration of 1 ~Ci/M1. Slides were processed for autoradiography [11] and the fraction of cells in S determined on at least 1000 cells. The peroxidase reaction was determined by the method of Hayhoe [12], the Sudan black reaction by the method of Sheehan and Storey [13], and the periodid acid Schiff(PAS) reaction by the method of Hayhoe et al. [12]. Positive peroxidase reactions were considered those where weak diffuse granules or a tinge confined to Auer rods were present in the blast cells; positive Sudan black reactions those where blast cytoplasm showed variable amounts of sudanophilic granules or else only Sudan positive Auer rods; positive PAS reactions those where there was a crown of cytoplasmic granules or a single block of glycogen even if this particular type of staining reaction was present in only a very small number of immature cells. On the contrary, if blast ceils were completely negative or if they showed a weak diffuse tinge or, more exceptionally, if their cytoplasm contained PAS positive Auer rods, PAS reactions were considered negative [12]. RESULTS
The main features of the cases used for the present study are listed in Table 1. Leukaemias were classified as lymphoblastic or myeloblastic according to the following criteria of recognition. The group of lymphoblastic leukaemias included those with a high nucleocytoplasmic ratio, absence of Auer rods and of
Deoxycytidylate Deaminase Activity
613
Table 1. Clinical and cytochemieal data of the acute leukaemia cases
Case No. Acute myeloblastic ]eukaemia
Acute lymphoblastie leukaemia
Sex
WBC/ cumm x 10 a
% of blasts
Sudan black
Peroxidase
PA S
6 22 I0 70 70
F F M F F
66 274 14 184.6 15
90 87 84 86 67
+ + + + +
+ + + + +
------
75 6 4 45 65 4
F F M M F F
51 40 16 16 171 235
85 97 86 93 84 93
-------
-------
+ + + + + +
Patient
Age . (years)
1 2 3 4 5
A.P. C.V. S.E. R.A. C.M.
6
P.F. A.V. C.P. R.M. R.A. M.O.
7 8 9 10 11
other intracellular structures, negative peroxidase and Sudan black reactions and positive PAS reaction. T h e myeloblastic forms were considered those with a lower nucleo-cytop l a s m i c r a t i o , p r e s e n c e o f A u e r rods a n d / o r o f azurofil granulations, positivity of peroxidases and Sudan black reactions and negativity of PAS reaction. T h e results o f t h e d e t e r m i n a t i o n o f d C M P d e a m i n a s e a c t i v i t y a n d o f t h e effect o f a c t i n o m y c i n D u p o n the e n z y m e activity are shown i n T a b l e 2. T h e v a l u e s o f t h e a v e r a g e l a b e l l i n g i n d e x o f t h e b l a s t cells p o p u l a t i o n o f e a c h case a r e also r e p o r t e d . T h e effect o f a c t i n o m y c i n D is s h o w n i n t h e t a b l e as t h e p e r c e n t a g e o f i n h i b i t i o n o f e n z y m e a c t i v i t y , t a k i n g as 100 t h e enzyme activity of the untreated cultures. The effectiveness o f t h e dose e m p l o y e d w a s c h e c k e d
Table 2.
by its ability to reduce uridine incorporation into RNA. In both lymphoblastic and myeloblastic leukaemias actinomycin D inhibited uridine incorporation more than 90%. Furthermore, under these conditions, actinomycin D did not produce any gross morphological change in the cells nor differences in the cell viability, as judged by the trypan blue exclusion test, between treated and control cultures. Table 2 shows that the dCMP deaminase activity is significantly (P <0-05) higher in AML than in ALL cells. In addition, a close correlation was observed between dCMP deaminase activity and the percentage of labelled cells in the AML forms (Fig. 1). Enzyme activity was directly proportional to the proliferative activity. On the contrary, in acute
Deoxycytidylate deaminase activity and inhibition of the enzyme activity following actinomycin D treatment *mgmoles of dCMP deaminated/min/ % of 1"Labelling Case mg of protein inhibition indices
Acute myeloblasdc leukaemia
Acute lymphoblastic leukaemia
1
13.0+
2 3 4 5
20.8 + 0.7 27.4+ 0.1 35.9 + 19-4 48.6+ 7.1
6 7 8 9 10 11
24 + 23.6 + 4.8 + 2.5 + 12.4+ 9.4+
1.
1.3 3.5 2.5 1.1 0.3 0.3
0 0 0 4
2.6 3.1 3.7 4.3 5
73 68.5 68 42 14 10-3
1 2.4 2"2 9-8 18.4 19.6
0
*Each number is the average of three determinations. ~Percentage of 3 H - T d R labelled blast cells.
Maria Grazia Bernengo and Luigi Pegoraro
614
lymphoblastic leukaemia d C M P deaminase activity exhibited a greater range of variability and was not related with the cell proliferative rate. The effect of actinomycin D upon the activity of the enzyme revealed different behaviour pattern between the two types of leukaemias, d C M P deaminase activity in all cases of A M L was not affected by the 9-hr actinomycin D treatment, while in all cases of ALL a decrease of enzyme activity was ob-
$
~ ..4 z_.
-t m
2
,J
0
m
I
I
20
I
I
40
I
13"11.1 MOLES dCMP DEAMINATED//MIN/MG
I
60
OF PROTEIN
Fig. 1. Correlation between the percentage of labelled blast cells (ordinate) and the dCMP deaminase activity (abscissa), expressed in mltmoles deaminated/minute[mg of protein, in the A M L cases.
I
I 3
% OF INHIBITION
Fig. 2. Correlation between the percentage of labelled blast cells (ordinate) and the percentage of inhibition of the dCMP deaminase activity by actinomycin D (abscissa) in the ALL
cases. (u - - 0"15%).
served. Since the degree of inhibition varied from case to case a possible relationship of the actinomycin effects with the proliferative activity of the ALL cells was investigated. A close correlation (Fig. 2) was observed between the template stability and the labelling indices.
DISCUSSION The results of this study show that the blast cells o f A M L and of ALL possess a different ability to deaminate dCMP. In fact, the activity of d C M P deaminase was found to be higher in the myeloblastic than in the lymphoblastic forms in spite of the fact that the latter possess a higher proportion of cells in the S phase of the cell cycle. In A M L a direct correlation was found between the proliferative activity and the enzyme activity of the blast cells. Increased activity of some of the enzymes of the DNA synthetic pathway, including d C M P deaminase have been shown to occur preceding or concomitantly with DNA synthesis in a number of systems [14, 15]. It is not surprising therefore that those A M L cases with a higher fraction of cells in S and hence, with a higher demand of T M P synthesis, also possess a higher d C M P deaminase activity. It is not clear, however, why in the ALL forms such correlation is absent. It is possible that this represents an imbalance in the regulation of the enzyme molecules synthesis or activity. However, one must also consider the possibility that the group of ALL is heterogeneous in the sense that m a n y subgroups of ALL are included in it. Cytochemical investigations seems to support this view [16]. The main difference found in the present study between the A M L and ALL forms concerns the stability of the enzyme activity to actinomycin D treatment. Actinomycin D, at the dose we used, almost completely inhibits R N A synthesis, in both A M L and ALL cells, and hence prevents the synthesis of new templates. It is therefore likely that a decrease of d C M P deaminase activity in the treated cultures is due to depletion of the RNA templates specific for this enzyme. The fact that after 9 hr of actinomycin D treatment the activity of d C M P deaminase in A M L cells it is unchanged while in ALL cells it is strongly decreased suggests that the former possess a more stable template for this enzyme. The stability of the templates of the enzymes of the DNA synthetic pathway has been extensively studied, with the aid of actinomycin D, both in systems of stimulated DNA synthesis and in continuously
Deoxycytidylate Deaminase Activity
dividing normal and neoplastic cells. In quiescent or Go cells RNA templates were found, in most instances, to be more stable than in cells stimulated to undergo to DNA synthesis [1721]. We have shown that the treatment of P H A stimulated normal lymphocytes with actinomycin D for 9 hr preceding the maximum DNA synthesis decreases to 50% of the d C M P deaminase activity. Conversely, in unstimulated lymphocytes, treatment for 9 hr with actinomycin D does not affect the enzyme activity [22]. The stability o f d C M P deaminase activity to actinomycin D treatment has also been found to be greater in normal than in regenerating liver [17]. In rapidly proliferating normal and neoplastic cells little or no effect of actinomycin D has been shown upon the activity of the enzymes of the DNA synthetic pathway [23-25]. In the present study A M L cells, when treated with actinomycin D, behave in the same way as Novikoff hepatoma and Ehrlich ascites cells, while, in most cases, ALL cells are very similar to cells stimulated to DNA synthesis. Furthermore, in the A L L a correlation between the d C M P deaminase template lability and the proliferative activity of the blast cells was found, suggesting that the half fife of the
R N A templates is directly proportional to the percent of cycling cells. Since the enzyme level within a cell is dependent not only upon the stability of its templates but also upon the turnover rate of the enzyme protein itself, the difference between A M L and ALL ceils could depend upon a different half life of the d C M P deaminase molecules. The fact that other systems such as normal adult and regenerating fiver possess equally stable thymidine kinase but different turnover for the template of this enzyme may suggest that also A M L and ALL cells have a similar pattern [26]. Unfortunately, our attempts to evaluate the half life of d C M P deaminase with cycloeximide treatment in acute leukaemic cells were unsuccessful. We cannot therefore assert from our results whether the difference between the ALL and the A M L cells lies at the level of transcription or at the level of degradation of the enzyme molecules. Our data concerning the d C M P deaminase have shown differences between A M L and ALL both in the activity of the enzyme and in effects of actinomycin D upon this activity. It may be worth further studies to see whether this difference is limited to d C M P deaminase or is it common to other enzymes of the DNA synthetic process.
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