[CANCER RESEARCH 42, 1326-1330, April1982]0008-5472/82/0042-0000$02.00

Furine and Pyrimidine Ribonucleoside Monophosphate Patterns of

Peripheral Blood and Bone Marrow Cells in Human Acute

Leukemias

JoëlleScavennec,1 Dominique Maraninchi, Jean-Albert Gastaut, Yves Carcassonne, and HélèneL. Cailla

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9 [J. S., H. L. C.¡.and Institut J. Paoli I. Calmettes, 232', bd de

Sainte-Marguerite, BP 156. 13273 Marseille Cedex 9 [D. M., J-A. G., Y. C.], France

ABSTRACT

Purine and pyrimidine ribonucleoside monophosphate (NMP)levels were measured in blood and/or bone marrow cells cfgroups of patients with acute leukemia including: (a) acutelymphoblastic leukemia (ALL); and (b) acute nonlymphoblasticleukemia (ANLL) (acute myeloblastic, promyelocytic, myelo-

monocytic, and monoblastic leukemia). Patients were eitheruntreated or in remission (17 with ANLL and six with ALL);three relapsed patients with ALL were also studied. Phytohe-magglutinin-stimulated tonsil lymphocytes and bone marrowcells both from normal subjects were used as controls. Moreover, observations in acute leukemias were compared withthose in chronic leukemias.

The cytidine, adenosine, guanosine, and uridine 2'-, 3'-, and5'-monophosphate contents were measured by high-perform

ance liquid chromatography. Using this technique, relativelydifficult separations (e.g., adenosine of 2'-, 3'- and 5'-mono-

phosphates) were achieved fairly rapidly by isocratic elution.2'- and 3'-NMP concentrations were 2.5 times higher (0.69 to1.38 x 10~9 mol/106 cells in the peripheral blood and 2.42 to5.87 x 10~9 mol/106 cells in the bone marrow) in ALL andwere 5.5 times higher (1.66 to 3.82 x 10~9 mol/106 cells inthe peripheral blood and 4.30 to 9.27 x 10~9 mol/106 cells in

the bone marrow) in ANLL leukemic cells than in chronicleukemic blood cells or control bone marrow cells (0.34 to0.75 and 0.93 to 1.63 x 10~9 mol/106 cells, respectively). 2'-

NMPs were found only in leukemic blast cells and accountedfor about 30% of 2'- and 3'-NMP mixtures. Levels of 2'- and3'-NMP were around 2- and 4-fold higher in bone marrow thanin peripheral blood cells of patients. The high level of 2'- and3'-NMP found in bone marrow and blood cells of untreated ALL

patients decreased during remission without reverting to normal; however, in relapse, it was 5 to 10 times higher than inthe case of untreated patients.

The 5'-NMP concentrations were always lower than those of2'- and 3'-NMP (e.g., cytidine 5'-monophosphate = 0.29 x10~9 mol/106 cells and cytidine 2'- and 3'-monophosphates= 6.40 x 10~9 mol/106 cells in bone marrow of untreated

patients with ANLL) and were the same in acute leukemias andcontrol (including chronic leukemias). Furthermore, 5'-NMPlevels remained constant during the course of the disease. 2'-and 3'-NMP levels appear therefore to be useful markers of

leukemic blasts and may facilitate both the diagnostic of acuteleukemia and the monitoring of chemotherapy.

INTRODUCTION

Levels of nucleosides and nucleotides in human leukemiccells have been investigated by several groups. Bishop ef al.(2), Willoughby and Waisman (24), and Ondarza (16) havecompared normal and leukemic whole blood. The followingcompounds were detected: di- and triphosphopyridine nucleotides, uric acid, AMP, ADP, ATP, UDPAG,2 UDPG, GTP, and

DTP. Nakai ef al. (15) later identified and quantified the majoracid-soluble nucleotides in normal and leukemic human leukocytes: di- and triphosphopyridine nucleotide, CMP, CDP,

CTP, UMP, UDP, UTP, GMP, GDP, GTP, AMP, ADP, ATP,UDPAG, and UDPG. UDPG and UDPAG were present in somewhat greater quantities in leukocytes from patients with acutemyeloblastic and chronic myelocytic leukemia. A similar studyperformed recently in rat hepatomas by Jackson ef al. (11)indicated that the patterns of pyrimidine and purine nucleotidelevels can be correlated to transformation and progression inhepatomas.

Moreover, several investigators detected only quantitativedifferences in the activities of the different enzymes involved innucleotide synthesis and metabolism in normal and leukemiccells. The initial observations by Beck and Valentine (1 ) showedthat leukocytes of patients with chronic myelocytic leukemiahave diminished or undetectable alkaline phosphatase activity.Hande and Chabner (8) found higher enzyme levels of pyrimidine nucleoside monophosphate kinase in human leukemicblast cells than in normal blast cells. High activities of DNApolymerase and terminal deoxynucleotidyltransferase havebeen reported in untreated or relapsed leukemic patients withALL (18).

Our previous studies (19) suggested that high cyclic cytidine3':5'-monophosphate concentrations in peripheral blood cells

are characteristic of acute leukemias, whether myeloblastic,monoblastic, or lymphoblastic. In contrast, cyclic cytidine 3':5'-monophosphate levels were low in leukocytes from chronic

leukemias and undetectable in leukocytes from controls. Lowcyclic adenosine 3':5'-monophosphate concentrations were

clearly associated with leukemic disease independently of thelineage of the affected cell or its stage of differentiation. Cyclicguanosine 3':5'-monophosphate levels were not significantly

different in leukemic or control cells. These results confirm andextend those already reported by Peracchi ef al. (17).

We now report observations on noncyclic CMP, AMP, GMPas well as UMP. HPLC greatly facilitates analysis of the 2', 3',

' To whom requests for reprints should be addressed.

Received April 8. 1981; accepted December 9, 1981.

2 The abbreviations used are: UDPAG, uridine diphosphoacetylglucosamine;

UDPG, uridine diphosphoglucose; ALL, acute lymphoblastic leukemia; HPLC,high-pressure liquid chromatography; NMP, ribonucleoside monophosphate;ANLL, acute nonlymphoblastic leukemia; PHA, phytohemagglutinin A.

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r;:

and 5' isomers of these NMPs and allows a comparative study

of their cellular levels. Untreated patients with ANLL and ALLwere studied as well as patients in remission and in relapse.

The NMP contents of leukemic blasts were compared tothose of leukocytes from patients suffering either from chronicmyelocytic leukemia or chronic lymphocytic leukemia. PHA-stimulated tonsil cells and normal bone marrow cells were usedas controls.

MATERIALS AND METHODS

Chemicals. Furine and pyrimidine NMPs were purchased fromSigma Chemical Co. St. Louis, Mo. Ficoll 400 and Telebrix 38 wereobtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and Laboratoire Guerbet, Aulnay-sous-Bois, France, respectively. Roswell

Park Memorial Institute Tissue Culture Medium 1630 and PHA, M form,were obtained from Gibco, Paisley, United Kingdom. [3HJThymidine (1

Ci/mmol) was purchased from the CEA, France.Instrumentation. HPLC analysis was performed on a liquid chro-

matography equipped with a Model U6K injector (Waters Associates,Milford, Mass.), a Pye Unicam LC-UV detector with variable wavelength

(Pye Unicam, Ltd., Cambridge, United Kingdom), and a reciprocatingpump "A," 350 bars (Oosapro Milton Roy, Port-Saint-Pierre, France).

A 10-mV W + W Recorder 600 Tarkan was used. Separations werecarried out on a weak anion-exchanger NH;,-uBondapak column [30

cm x 3.9 mm (inside diameter)] from Waters Associates.Patients. There were exclusively adult patients, male or female,

hospitalized at the Institute J. Paoli I. Calmettes. Leukemia diagnosiswas established by complete hematological and cytological examination. The clinical features of each patient are presented in Table 1.Peripheral blood and bone marrow (taken by posterior iliac crestaspiration) were drawn at the same time except when indicated (see

NMPs in Human Acute Leukemias

Table 1). Of the 17 patients with ANLL, 9 were untreated and 8 werein hematological remission. Of the 9 patients with ALL, 3 were untreated, 3 in remission, and the other 3 in relapse. Five normal bonemarrow specimens were analyzed as controls. Tonsils from 2 childrenundergoing tonsillectomy for benign disease were used as a source ofPHA-stimulated lymphocytes. Furthermore, blood samples from 5 pa

tients suffering from chronic leukemias were taken for the purpose ofadditional comparisons.

Leukocyte Preparation. Peripheral blood (50 ml) and bone marrow(2 ml) were drawn into heparinized syringes and treated within 2 hr.Leukocytes were purified on Ficoll density gradient according to Boyum(3). Cell viability, assessed by trypan blue dye exclusion, was alwaysgreater than 95%. These preparations usually contained less than 10%erythrocytes and a neglible number of platelets, comprised of up to80% leukemic blasts (acute leukemia), 20% blasts (normal bone marrow, the rest being mainly lymphocytes), and 0% blast (chronic leukemia). Cells obtained from surgically removed tonsils were resuspended(106 cells/ml) in Roswell Park Memorial Institute Tissue Culture Me

dium 1630 supplemented with 10% fetal calf serum and antibiotics, inthe presence of 1 fig PHA per ml. Cell proliferation was measured by[3H]thymidine incorporation. After 2 days of culture, 80% were lympho-

blasts as estimated by cytofluorography (13). Cells were harvested atthis stage; the viability was about 70%. Following purification, cellswere washed twice in Hanks' balanced salt solution, taken up at 5 x

107 cells/ml in 1 N HCIO4, and stored at -80°.

NMP Analysis. After thawing, extracts were centrifugea to removeprecipitated protein. The supernatants were neutralized with 9 N KOHand centrifugea to remove the precipitate. The neutral supernatantswere filtered through a Millipore filter (HAWP 0.45 ftm) (Millipore,Molsheim, France), and aliquots of these samples corresponding to105 to 10e cells (usually 25 jul) were analyzed by HPLC, under isocratic

conditions, on a weak anion-exchanger column equilibrated and eluted

with 0.01 M potassium phosphate, pH 3. Buffer conditions were as

Table 1Main clinical and hematological data shown by patients at the time of study

BloodWBC(x

10VBlastsPatientNamecode1

A.F.2M.A.3

O.P.4B.C.5

Y.K.6C.0.7S.M.8

P.S.9P.M.10

M.A.11A.M.12D.Y.1

3 B.Co.14G.J.1

5 Y.K.16Y.K."17

Y.K.18A.Ma.19V.A.20D.J."21

T.H.22B.G.23G.C.24E.F.25P.E.26M.An.'*

+ + + + , very hypercellular; + + HSexFMMMMFFFFMFFFFMMMFFMMMFFMFAge

(yr)Diagnosis5956826540287075465629411742404040576158742131195521ANLLRem.c

ANLLALL8ALL9ALL8Rem.

ALL9Rem.ALL9Rem.ALL9Rei.ALL8Rei.ALL9Rei.

ALL9cu

mm)(%)26.68.428.89.232.23.124.462.919.67.010.011.56.93.60.81.65.42.26.860.04.213.413.411.24.110.6-,

hypercellular; + + , moderately hypercellular; -,757365508838909493000000004082950006506normocellular.Bone

marrowBlasts(%)

Richness60+ +++*90

++70+ ++54+ ++80+ + ++81++100+ + ++76+ ++70+ + ++12

+ ++1++3++1++142

+ + ++98+ + ++97+ ++94+ + ++00

+ + ++1+ ++33+ ++66+ + ++85+ + + +Time

ofre-Treatmentmission(wk)b——————-_+

32+++4.+

3+5+

23__+4.++++"

—, without chemotherapy; + , withchemotherapy.cRem., remission; Rei.,relapse."Only bone marrow wastested.8ALL with nullcell.'Only peripheral blood wastested.aThe immunological characterization was not performed.

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J. Scavennec et al.

described by Edelson et al. (6) with a flow rate of 1.4 ml/min. Theelution was monitored at 260 nm. Peak areas were integrated, and theamounts calculated using standard NMPs at 23°were as follows: 5'-CMP, 3.12 min; 2'-CMP, 3.36 min; 3'-CMP, 3.48 min; 5'-AMP, 5 min;2'-AMP, 6.18 min; 3'-AMP, 6.54 min; S'-UMP and 5'-GMP, 12.1 2 min;2'- and 3'-UMP, 14.18 min; 2'- and 3'-GMP, 16 min.

Identification of NMPs Present in Blood and Bone Marrow Leukocytes from Leukemic Patients. NMPs were identified by their retention times and cochromatography with reference compounds. Theeluted peaks of cell extracts were identified in several ways: (a) analysisof UV spectra; (b) by including internal standards; and (c) by comparison with standard chromatograms.

Separation of 5' isomers from 2' and 3' isomers was readily accomplished, whereas the separation of 2' and 3' isomers was more difficult.

Under the Chromatographie conditions used, complete resolution of2'-, 3'-, and 5'-AMP was achieved; whereas for other NMPs, 2' and 3'

isomers were eluted, partially separated, in one peak. Attempts toachieve better separations of 2'- and 3'-CMP, 2'- and 3'-UMP, and 2'-and 3'-GMP by varying the pH of the buffer from 2.25 to 7 wereunsuccessful. The 2' isomer extent, estimated both in a 2'- and 3'-CMPmixture and in a 2'- and 3'-AMP mixture, was about 36 and 30%,respectively, in blood and bone marrow blasts. In control cells, 2'

isomers were below the detection threshold.Statistics. The differences in the whole NMP content (2'-, 3'-, and

5'-NMP) between the untreated ANLL and the control groups wereanalyzed using the Fisher's discriminative analysis. In this test, values

with 7 of the difference >2 were significant. The differences in 2'- and3'-NMP levels between the ANLL (untreated and treated) and the

control groups were analyzed by analysis of variance. Values with p< 0.05 were significant.

RESULTS

NMP Levels of Peripheral Blood WBC from Leukemic Patients. In Table 2, the NMP contents of leukemic blood blastsare compared with those of leukocytes from patients withchronic leukemias.

Among each group of patients, all the 2' and 3' isomers onthe one hand and all the 5' isomers on the other hand fell withinthe same concentration range. 2'- and 3'-NMP levels were 10times higher than those of 5'-NMP with one exception for 5'-

AMP in treated ANLL.2'- and 3'-NMP concentrations were always higher in the

peripheral blood WBC of patients with acute leukemias than inthose of patients with chronic leukemias; they were 5.5-foldhigher in ANLL (p < 0.05, Student's f test) and 2-fold higherin ALL. 5'-NMP levels did not show a significant difference

between acute and chronic leukemias.The whole NMP content of the ANLL group was compared

with the whole NMP content of the chronic leukemia group byFisher's discriminative analysis. These differences were highly

significant (T = 8.46).NMP Levels in Bone Marrow Cells. Table 3 shows NMP

levels of leukemic and normal cells from bone marrow. 2' and3' isomer concentrations in bone marrow cells were about 2

times (for ANLL patients) and 4 times (for ALL patients) higherthan in blood cells. As in the peripheral blood WBC, differencesbetween 2'- and 3'-NMP levels of ANLL and normal bone

Table 2

NMP contents of peripheral blood WBC from untreated and treated patients with ANLL or ALL and of WBCfrom chronic lymphocytic and chronic myelocytic leukemia patients

Extraction and analysis procedures were as described in "Materials and Methods."

Untreatedpatients5--CMP

2'- and 3--CMP5--AMP2'- and 3--AMPS'-UMP and 5M3MP2'- and 3MJMP2'- and 3--GMPANLL

(n =9)0.20±0.266

3.09 ±1.72°

0.60 ±0.352.68 ±1.52C

0.30 ±0.151.66 ±1.05C3.82 ±2.20°ALL

(n -2)0.07

±0.021.03 ±0.240.14 ±0.170.95 ±0.540.24 ±0.170.69 ±0.261.38 ±0.77Treated

patients (inremission)ANLL

(n =6)0.21

±0.201.47 ±1.28*2.29 ±3.83d1.95 ±1.08d

0.63 ±0.670.93 ±0.84d2.47 ±1.67aALL

(n =3)0.07

±0.050.30 ±0.170.17 ±0.030.35 ±0.250.12 ±0.160.26 ±0.200.57 ±0.54CLLfl

(n - 3)

and CML(n = 2)

patients0.05

±0.040.50 ±0.84s

0.06 ±0.040.50 ±0.858

0.03 ±0.020.34 ±0.62e0.75 ±1.38e

" CLL, chronic lymphocytic leukemia; CML, chronic myelocytic leukemia.A Mean ±S.D. of n determinations x 10~9 mol/106 cells.c p< 0.05 compared to chronic lymphocytic and chronic myelocytic leukemia patients (Student's t test).d p £0.20 compared to untreated patients with ANLL (Student's f test).9 p s 0.05 compared to patients with ANLL (untreated and treated) (analysis of variance).

Table 3

NMP contents of bone marrow leukocytes from untreated and treated patients with ANLL or ALL and ofnormal bone marrow leukocytes

Extraction and analysis procedures were as described in "Materials and Methods."

UntreatedpatientsS'-CMP

2'- and3'-CMP5'-AMP

2'- and3'-AMP5'-UMP and S'-GMP

2'- and 3MJMP2'- and 3'-GMPANLL

(n -9)0.29±0.17"

6.40 ±4.811.01±1.33

5.42 ±3.79b0.59

±0.294.30 ±2.91*9.27 ±6.43"ALL

(n =2)0.24

±0.273.98 ±2.351.21

±0.164.28 ±0.510.39

±0.022.42 ±1.225.87 ±2.10Treated

patients (inremission)ANLL

(n =9)0.28

±0.233.00 ±2.50°2.47

±4.833.30 ±2.16C1.18

±1.432.41 ±1.56C5.40 ±2.59CALL

(n =2)0.09

±0.071.13 ±0.380.13

±0.121.28 ±0.600.22

±0.261.05 ±0.252.61 ±1.18Normal

bonemarrow(n

=5)0.08

±0.081.47 ±1.29d0.09

±0.061.04 ±0.70d0.80

±0.660.93 ±0.65o1.63 ±1.26"

" Mean ±S.D. of n determinations X 10 9 mol/108 cells.0 p £0.05 compared to normal bone marrow (Student's i test).c 0.10 < p < 0.20 compared to untreated patients with ANLL (Student's ( test).d p £0.05 compared to patients with ANLL (untreated and treated) (analysis of variance).

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NMPs ¡nHuman Acute Leukemias

marrow cells were highly significant both for 2'- and 3'-NMPtaken independently ( p s 0.05, Student's f test) and for all the2', 3', and 5' isomers taken together (T = 5.07).

In PHA-stimulated lymphocytes, NMP concentrations(X10~9 mol/106 cells) were as follows: 5'-CMP, <0.01; 2'-

and 3'-CMP, 0.4; 5'-AMP, 0.11 ; 2'- and 3'-AMP, 0.97; 5'-UMPand 5'-GMP, 0.18; 2'- and 3'-UMP and 2'- and 3'-GMP levels

were too close to the background to be accurately measured.These values, when compared to the NMP concentrationsfound in blood and bone marrow leukemic blasts, were in thesame range as or even lower than those of the peripheral bloodWBC from chronic leukemia.

Evolution of Patterns of NMP According to Disease Stagein ANLL and ALL. The question arises as to whether the NMPcontent of leukemic blasts changes simultaneously to remissionand relapse.

Tables 2 and 3 show the NMP levels of the peripheral bloodWBC and bone marrow leukocytes from untreated and treatedpatients with acute leukemia. Analysis of the results corresponding to ANLL revealed that the 2'- and 3'-NMP levels in

cells from patients in remission were one-half of those in blasts

from untreated subjects but were still higher than in WBC fromchronic leukemia and normal controls (PHA-stimulated lympho

cytes and normal bone marrow cells). The difference betweenvalues of untreated and of treated patients with ANLL was notsignificant (p < 0.20, Student's t test).

The 5'-NMP concentrations in the blasts from untreated

patients were roughly the same as in cells from patients inremission, with the exception of 5'-AMP which increased in the

latter.We also compared the NMP contents in peripheral blood

leukocytes and bone marrow cells from ALL patients. As shownin Tables 2 and 3, the 2'- and 3'-NMP levels were 2.5-fold

•12S"o

8{'•

o; non,r„nñ-TIIiFISXaUP Z3CMP 5-AMP Z3BMP SUMP 23UMP 23<GMP

5GMP

.ILr' Jm-PlludPli1

5'CMP 23CMP 5'AMP 2?'AMP 5\JMP OUMP 23GMP

5GMP

Chart 1. Concentrations ot purine and pyrimidine NMPs in leukocytes frompatients with ALL untreated, in remission, and in relapse. Leukocytes isolatedfrom peripheral blood and bone marrow were acidified by HCIO., and neutralizedby KOH. NMP contents were analyzed by HPLC, as described in "Materials andMethods." Top, bone marrow; a, untreated patients (n = 2); b, patients inremission (n = 3); c, relapsed patients (n —3). Bottom, peripheral blood; a,untreated patients (n = 2); b, patients in remission (n = 2); c. relapsed patients(n = 2). Data are expressed as x 10~9 mol/106 cells.

lower in cells from ALL patients in remission than in blasts fromuntreated patients.

In Chart 1, the evolution of the NMP contents with the stageof ALL is shown. Both in blood and bone marrow cells, thehighest NMP levels were observed in patients in relapse whilethe lowest were observed in patients in remission; they were,however, never as low as in controls. From remission to relapse, the NMP increases ranged from 5-fold in bone marrowcells to 10-fold in peripheral blood leukocytes.

DISCUSSION

In all cases of untreated patients with acute leukemias (ANLLor ALL), the concentrations of purine and pyrimidine NMPs,especially of the 2' and 3' compounds, were significantly higher

in leukemic blasts than in control cells. The choice of appropriate controls for leukemic blasts is still an unresolved problemsince enough separated normal human lymphoblasts or mye-

loblasts for biochemical studies cannot be obtained. However,our main concern was defining the nucleotides of acute leukemic blasts and the possible clinical usefulness of such measurements. For comparison of leukemic blast cells, we usednormal lymphoblasts obtained by PHA stimulation of normallymphocytes and leukocytes from normal bone marrow andfrom blood of chronic leukemic patients. Although there areobviously not ¡dealsystems, these "controls" with 0% (chronic

leukemic cells), 20% (normal bone marrow), and 80% (PHA-

stimulated lymphocytes) show very low NMP levels.NMP levels were higher in acute leukemic blasts than in

chronic leukemic leukocytes, indicating that mature leukemiccells do not display the same NMP pattern as do immatureleukemic cells. On the other hand, NMP levels were also higherin leukemic bone marrow blasts than in normal bone marrowblasts, supporting the hypothesis that the peculiar NMP levelsare not due to the immaturity of these cells but rather to somealteration in the differentiation of leukemic cells.

The large standard deviations we observed are not surprisingsince it has been reported that, during leukemic blast maturation, some cells proliferate while others do not and may evendie at any stage of their development (12, 23). The blastpopulation in a given patient, particularly in ANLL, cannottherefore be considered as homogeneous (7, 12, 14). Nevertheless, the differences found between ANLL patients andcontrols were statistically significant.

The NMP concentrations in bone marrow blasts were 2- and4-fold higher than in peripheral blood blasts in ANLL and ALL

patients, respectively. The differences found between bonemarrow and blood could be attributed to the inability of peripheral blood to reflect entirely the proliferative activity of bonemarrow (9, 14).

When the repartition of the different isomers of NMP inleukemic blasts from untreated patients was estimated, theresults showed that the concentrations of 2'- and 3'-NMP werehigher than the concentrations of 5' isomers. Normal cells

showed a similar pattern but to a lesser degree. The estimationof the 2'-NMP contents of acute leukemic blasts indicated that2'-CMP and 2'-AMP represented 36 and 30%, respectively, ofthe 2' and 3' isomer mixture.

As far as we are aware, the presence of 2' isomers as normal

components of cells has never been described. The only dataon this subject show the existence of an enzyme in nervous

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J. Scavennec et al.

tissues which specifically cleaves cyclic ribonucleoside 2':3'-monophosphate into 2'-NMP (5, 10). However, until now, therewas still no evidence that cyclic ribonucleoside 2':3'-mono-

phosphate is a natural substrate for this enzyme. A tentativeexplanation for the presence of 2' isomers would be the exis

tence of such an enzyme in leukemic blasts.What is the origin of the excess of NMP in acute leukemic

blasts? It has been verified that these data could not beattributed to RNA degradation during the processing of thecells. Cultured cells have been analyzed: PHA-stimulated lym

phocytes and 3 lymphoblastoid cells (Raji, Molt 4, and Ramoscells), for which a purification on Ficoll density gradient is notnecessary. While PHA-stimulated lymphocytes have a low NMP

content, the lymphoblastoid cell lines have a NMP content inthe same range as those of leukemic blasts. Because purineand pyrimidine NMPs, particularly 3' isomers, vary by about

the same extent and in the same sense, some disturbance inRNA degradation which normally produces the 3'-NMP can be

postulated. Several data are available about RNA metabolismin leukemic cells (4, 20, 21 , 22), but none deal with the possibleaccumulation of degradation products. This will be the subjectof a future study.

Our results show a further dissimilarity between the NMPcontents of cells from untreated patients and from patients inremission or in relapse. Levels of 2'- and 3'-NMP were de

creased in cells from patients in remission but still higher thanin controls. Their concentrations were highest in patients inrelapse. Preliminary observations suggest that this elevationoccurs before relapse can be detected by morphological criteria. Studies of additional patients are being undertaken toconfirm these findings. If they prove to be reproducible, auseful early marker of relapse will become available.

ACKNOWLEDGMENTS

The authors wish to thank Or. J. Fondami (Institut J. Paoli I. Calmettes) whoperformed statistical analysis of our results and Or. A. C. Allison for his commentsand advice.

REFERENCES

1. Beck, W. S., and Valentine, W. N. Biochemical studies on leukocytes. II.Phosphatase activity in chronic lymphatic leukemia, acute leukemia, andmiscellaneous hématologie conditions. J. Lab. Clin. Mod , 38: 245-253,1951.

2. Bishop, C., Rankine. C. M., and Talbott. J. H. The nucleotides in normalhuman blood. J. Biol. Chem., 234. 1233-1237, 1959.

3. Boyum, A. Separation of leukocytes from blood and bone marrow. Scand.J. Clin. Lab. Invest. Suppl., 97: 1-108. 1968.

4. Cline, M. J. Metabolism of RNA in normal and leukemic cells. Acta Haematol.(Basel). 45: 174-180, 1971.

5. Drummond, G. I., Iyer, N. T., and Keith, J. Hydrolysis of ribonucleoside 2',3'-cyclic phosphates by a diesterase from brain. J. Biol. Chem., 237: 3535-

3539, 1962.6. Edelson, E. H., Lawless, J. G., Wehr, C. T., and Abbott, S. R. Ion-exchange

separation of nucleic acid constituents by high-performance liquid chroma-tography. J. Chromatogr., 174: 409-419, 1979.

7. Greenberg, M. L. Cellular kinetics in the leukemias. In: A. Rubin and S.Waxman (eds.). The Leukemia Cell, p. 89. West Palm Beach. Fla.: CRCPress, 1979.

8. Hände, K. R., and Chabner, B. A. Pyrimidine nucleoside monophosphatekinase from human leukemic blast cells. Cancer Res.. 38: 579-585, 1978.

9. Hillen, H. P. F., Wessels, J. M. C., and Haanen. C. A. M. Principles of pulsecytophotometry and the application in DNA measurements. In: Cell Proliferation Patterns in Acute Leukaemia Monitored by Pulse-Cytophotometry.

p. 23. Ghent, Belgium: European Press Medikon, 1975.10. Hugli, T. E., Bustin, M., and Moore, S. Spectrophotometric assay of 2'.3'-

cyclic nucleotide 3'-phosphohydrolase: application to the enzyme in bovinebrain. Brain Res.. 58: 191-203. 1973.

11. Jackson, R. C., Lui, M. S., Boritzki, T. J., Morris, H. P., and Weber, G.Purine and pyrimidine nucleotide patterns of normal, differentiating, andregenerating liver and of hepatomas in rats. Cancer Res., 40: 1286-1291,1980.

12. Killmann, S. A. Proliferative activity of blast cells in leukemia and myelofi-

brosis. Morphological differences between proliferating and nonproliferatingblast-cells. Acta Med. Scand., 178: 263-280, 1965.

13. Malissen, B., Charmot, D., Liabeuf, A., and Mawas, C. Expansion of humanlymphocyte populations expressing specific immune reactivities. I. Differential effects of various lectins on the expression of alloreactive cytotoxicityby primed cells. Immunology, J23: 1781-1787, 1979.

14. Mauer, A. M., and Fisher, V. Comparison of the proliferative capacity ofacute leukaemia cells in bone-marrow and blood. Nature (Lond.), 193:1085-1086, 1962.

15. Nakai, G. S., and Craddock, C. G. Acid-soluble nucleotides in leukemiccells. Cancer Res.. 25: 575-578. 1965.

16. Ondarza, R. N. Free nucleotides in normal and leukemic blood cells. ActaPhysiol. Lat. Am., 10: 129-134, 1960.

17. Peracchi, M., Maiolo, A. T., Lombardi, L., Catena, F. B., and Polli, E. E.Patterns of cyclic nucleotides in normal and leukaemic human leucocytes.Br. J. Cancer, 41: 360-371, 1980.

18. Sahai Srivastava, B. I. Biochemical markers for hematopoietic cell differentiation and leukemias. In: F. Ahmad, J. Scnultz, T. R. Rüssel,and R. Werner(eds). Differentiation and Development: Miami Winter Symposia, Vol. 15, p.523. New York: Academic Press, Inc., 1978.

19. Scavennec, J., Blanc, A. P., Gastaut, J.-A., Carcassonne, Y., and Cailla, H.L. Relationship between the levels of cyclic cytldine 3':5'-monophosphate,cyclic guanosine 3':5'-monophosphate. and cyclic adenosine 3':5'-mono-

phosphate in urines and leukocytes and the type of human leukemias.Cancer Res., 41: 3222-3227, 1981.

20. Torelli, U., Torelli, G. M., Andreoli, A., and Mauri, C. Partial failure ofmethylation and cleavage of 45S RNA in the blast cells of acute leukemia.Nature (Lond.), 226:1163-1165, 1970.

21. Torelli, U. L., Torelli, G. M., Andreoli, A., and Mauri, C. Impaired processingof ribosomal precursor RNA in blast cells of acute leukemia. Acta Haematol.(Basel), 45. 201-208, 1971.

22. Torelli, U., Torelli, G., Cadossi, R., Ferrari, S., Ferrari, S., Narni, F., andMontagnani, G. Accumulation of giant heterogeneous RNA molecules inacute leukemia blast cells. Cancer Res., 36: 4631-4638, 1976.

23. Wagner, H. P., Cottier, H., and Cronklte, E. P. Variability of proliferativepatients in acute lymphoid leukemia of children. Blood, 39. 176-186, 1972.

24. Willoughby, H. W., and Waisman. H. A. Nucleic acid precursors and nucleotides in normal and leukemic blood. I. Comparison of formic acid chromat-ograms. Cancer Res., 17: 942-947, 1957.

1330 CANCER RESEARCH VOL. 42

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1982;42:1326-1330. Cancer Res   Joëlle Scavennec, Dominique Maraninchi, Jean-Albert Gastaut, et al.   Leukemiasof Peripheral Blood and Bone Marrow Cells in Human Acute Purine and Pyrimidine Ribonucleoside Monophosphate Patterns

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