Relationship Between the Levels of Cyclic Cytidine 3':5'-Monophosphate

Home , Cyclic nucleotide, Guanosine

[CANCER RESEARCH 41, 3222-3227, August 1981] 0008-5472/81 /0041 -OOOOS02.00 Relationship between the Levels of Cyclic cytidine 3':5'-Monophosphate, Cyclic Guanosine 3':5'-Monophosphate, and Cyclic Adenosine 3':5'- Monophosphate in Urines and Leukocytes and the Type of Human Leukemias1

JoÃ«lle Scavennec,2 Yves Carcassonne, Jean-Albert Gastaut, AndrÃ©Blanc, and HÃ©lÃ¨neL.Cailla3

Centre Â¿Immunologie INSERM-CNRS de Marsei/le-Luminy. Case 906, 13288 Marseille cÃ©dex9[J. S.. H. L. C.], and Clinique des Maladies du Sang, HÃ´pital de la Conception, 13005 Marseille [A. B., J-A. G., Y. C.], France

ABSTRACT Since then, specific cyclic CMP phosphodiesterase has been described (10, 13), but an enzymatic activity converting CTP Cyclic cytidine 3':5'-monophosphate (cyclic CMP), cyclic into cyclic CMP has not yet been clearly established. Little is guarÃosme 3':5'-monophosphate (cyclic GMP), and cyclic known about cyclic CMP itself because no quantitative assay adenosine 3':5'-monophosphate (cyclic AMP) contents of leu was available until we developed a sensitive radioimmunoassay kocytes and urines of leukemic patients have been investi for cyclic CMP based on our previous work with cyclic AMP gated. We have studied four types of leukemia: acute myelo- and cyclic GMP radioimmunoassays (6, 7). blastic leukemia; chronic myelocytic leukemia; acute lympho- This new tool enabled us to study the cyclic CMP content in blastic leukemia; and chronic lymphocytic leukemia. As con urine and blood cells of patients with leukemia in an attempt to trols, the cyclic nucleotide content of leukocytes and urines of correlate the cyclic CMP concentration with the type or the healthy volunteers and patients with solid tumors selected for stage of leukemia. their normal hemogram has been determined. It has also been The 4 types of leukemia analyzed were AML, CML, ALL, and measured in phytohemagglutinin-stimulated lymphocytes. CLL. Our data show that: (a) the concentration of cyclic CMP is Results were compared with those from healthy volunteers always lower than that of cyclic GMP or cyclic AMP; (b) in and patients with solid tumors but normal leukocyte counts, as urines, the concentrations of the three nucleotides are higher well as with those from PHA-stimulated lymphocytes. in patients than in healthy volunteers, the greatest differences Cyclic AMP and cyclic GMP were measured in parallel in being observed between the cyclic CMP concentrations of order to determine whether a relationship could be found acute leukemia patients and controls; and (c) in white blood between the concentrations of the 3 cyclic nucleotides (15). cells, cyclic AMP concentration is lower in leukemic than in normal cells. The cyclic GMP concentration is the same every MATERIALS AND METHODS where except in monoblastic cells and leukocytes from solid tumor patients. High cyclic CMP levels are associated only with Cyclic CMP and cyclic [3H] CMP (20 Ci/mol) were purchased acute leukemia, whether myeloblastic, monoblastic, or lympho- from Boehringer, Mannheim, Germany, and Radiochemical blastic, a fact which suggests that cyclic CMP could be a Centre, Amersham, United Kingdom, respectively. Their purity biochemical marker of hematopoietic stem cell malignancy. was checked by electrophoresis on fluorescent cellulose ace tate, thin-layer chromatography on silica, and column chro- INTRODUCTION matography on aniÃ³n exchanger; they comigrated in all these The ubiquity of cyclic AMP4 and cyclic GMP has long been systems. Cyclic AMP and cyclic GMP were purchased from Sigma Chemical Co, St. Louis, Mo. established, and both molecules have been implicated in the The antisera against cyclic AMP, cyclic GMP, and cyclic control of cell growth (11,14). More recently, a pyrimidine CMP and the corresponding labeled antigens ([125l]iodosucci- cyclic nucleotide, cyclic CMP, was postulated to play an im nyl cyclic nucleotide tyrosine methyl ester) were prepared in portant role in cell proliferation. Cyclic CMP was first described our laboratory as previously described (8). Ficoll 400 and by Bloch ef al. (4) as a pharmacological agent capable of Telebrix 38 were purchased from Pharmacia Fine Chemicals, reducing the lag time in the growth of L1210 cells when they Uppsala, Sweden, and Laboratoire Guerbet, Aulnay-sous-Bois, are shifted from unpermissive to permissive conditions. He also France, respectively. Roswell Park, Memorial Institute Medium reported the presence of cyclic CMP in L1210 cells, regener 1630 and PHA, M form, were obtained from Grand Island ating rat liver, and urine of patients with acute leukemia (2, 3). Biological Co., Paisley, United Kingdom. [3H]Thymidine (1 Ci/ ' This investigation was supported by INSERM Grant ATP 40.76.72. It was mmol) was purchased from CEA, Saclay, France. presented in part at the International Conference on Clinical Aspects of Cyclic Nucleotides, Vail. Colo., July 1979 (15). 2 This work is part of the ThÃ¨se de 3e Cycle, University of Aix-Marseille II. Treatment of Samples of Urine and Blood Supported in part by Groupements des Entreprises FranÃ§aises dans la Lutte contre le Cancer. Because of the rapid degradation of cyclic nucleotides in 3 To whom requests for reprints should be addressed. urine at room temperature and the impossibility of collecting 4 The abbreviations used are: cyclic AMP, cyclic adenosine 3':5'-monophos- 24-hr urine in many cases, the first urines of the day were phate; cyclic CMP, cyclic cytidine 3':5'-monophosphate; cyclic GMP, cyclic guanosine 3':5'-monophosphate; AML, acute myeloblastic leukemia; ALL, acute collected and were used for both cyclic nucleotides and cre- lymphoblastic leukemia; CML, chronic myelocytic leukemia; CLL, chronic lym atinine determinations. A 10-ml aliquot of urine was transferred phocytic leukemia; PHA, phytohemagglutinin A; AMONOL, acute monoblastic leukemia. to a glass tube and immediately acidified with concentrated Received July 2, 1980; accepted April 29, 1981. perchloric acid (final concentration, 1 N) to avoid any cyclic

3222 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1981 American Association for Cancer Research. Cyclic Nucleotides in Human Leukemias nucleotide degradation. It was then stored frozen at â€”30Â°.At fractions supposed to contain cyclic CMP were pooled, lyoph the same time, 300 ml of blood were drawn into a bottle ilized, and reconstituted in a minimal volume of distilled water. containing acidified citrate dextrose as anticoagulant. This blood sample was immediately transferred to the laboratory. Radioimmunoassays of Cyclic Nucleotides Leukocyte Purification The radioimmunoassay is based upon the competition be Samples were purified on Ficoll density gradient according tween the antigen and radioactive analog for binding to a to the method of B0yum (5) as follows. Aliquots (60 ml) of specific antibody. The labeled analog of cyclic nucleotide is t125l]iodosuccinyl cyclic nucleotide tyrosine methyl ester. The freshly drawn blood diluted in an equal volume of 0.9% NaCI solution were automatically distributed into 100-ml centrifuge cyclic nucleotide to be assayed is converted into a 2'O-succinyl tubes, and 25 ml of Ficoll 400 and Telebrix 38 solution (d = derivative, a modification which increases the sensitivity by 2 1.071 ) were added directly on the bottom of each tube. Tubes orders of magnitude since the succinyl cyclic nucleotide is the were then centrifuged either in a Mistral MSE or Son/all RC3 hapten used in the immunogen (6, 8). centrifuge for 15 min at 1200 x g and 20Â°. Succinylation of Cyclic CMP. The sample containing cyclic The interface was totally recovered. After being washed CMP was first made 0.67 N in KOH. Then, a 150-/ul aliquot of twice with Hanks' balanced salt solution, the cells were resus- the alkaline solution was combined with 6.15 mg of lyophilized pended in Hanks' balanced salt solution, and the number of succinic anhydride, and the mixture was vortexed vigorously. cells was determined from an aliquot using either a Coulter Under these conditions, the cyclic CMP of the sample was quantitatively converted into its 2'-O-succinyl derivative as has Counter or a hemocytometer. The cell viability estimated by the trypan blue exclusion procedure was always above 95% at this been shown previously for other cyclic nucleotides (6, 7). stage. The cell differential in the Ficoll gradient was checked Radioimmunoassay of Cyclic CMP. A 4-fold dilution of the on smears stained with May-GrÃ¼nwald-Giemsa according to succinylated solution was mixed with an equal volume of 125I- standard techniques. More than 90% of the WBC were re labeled antigen solution and dialyzed against a suitable dilution covered in the interface fraction, which usually contained less of antibodies, and the free and antibody-bound radioactivities than 10% erythrocytes and a negligible Â«numberof platelets. were determined at equilibrium (6, 8). The amount of cyclic Once its volume was known, the cell suspension was acidi CMP was calculated from a standard curve constructed with fied by addition of concentrated HCIO4 (final concentration, 1 authentic cyclic CMP. Care was taken that the dilution of cyclic N) and stored at -80Â°. CMP used for the standard curve had the same ionic compo sition (0.1 M potassium Buccinate and 0.01 M potassium per- Preparation of Cyclic Nucleotide-containing Extracts chlorate) as did the samples to be assayed in order to exclude any interference due to salts contained in the succinylation All operations were carried out at 4Â°;urine and WBC received mixture. Concentrations of 2'-O-succinyl cyclic CMP as low as identical treatments. After thawing, denatured protein were 10~11 M can be measured. discarded by a centrifugation for 10 min at 4Â°. The acidic Control. Two rabbit antisera developed against 2'-O-succinyl supernatant was neutralized by 9 N KOH, and the KCIO4 was cyclic CMP and having different cross-reactivities were system eliminated by a brief centrifugation. atically used in order to further restrict the possibility of mea As described previously (6), all these operations allow a suring a molecule other than cyclic CMP. Table 1 shows that complete recovery of the free cyclic nucleotides in the neutral Antiserum 1C has a high affinity for the succinyl link since it is supernatant. After determination of its volume, this solution 3 orders of magnitude higher for 2'-O-succinyl cyclic CMP than was frozen and lyophilized if necessary. Lyophilized residue for cyclic CMP itself. Its specificity for cytosine is not very was reconstituted in a minimal volume of distilled water, and pronounced since it cross-reacts with the other 2'-O-succinyl the residual KCIO4 was centrifuged out. In this way, all the samples contained cyclic nucleotides in 10~2 M KCIO4 (con cyclic nucleotides. Antiserum 4C by contrast is specific for the pyrimidine nucleus, since it does not discriminate very well centration which corresponds to the solubility coefficient of this between cytosine and uridine but has almost no cross-reaction salt in these conditions). with 2'-O-succinyl cyclic AMP or 2'-O-succinyl cyclic GMP. Cyclic CMP Purification Only values consistent for both antisera were considered as significant. Four ml of the neutral sample were loaded onto a Dowex 1- As a control, the same sample was always assayed without X2 formate column (200 to 400 mesh) (11x1 cm), equilibrated, prior succinylation. To maintain the same ionic composition, a and eluted with 5 x 10~2 N formic acid at room temperature. second 150-/J aliquot of the alkalinized solution was pipetted CMP was first to elute followed by cyclic CMP; the other cyclic onto 7.1 mg lyophilized succinic acid and then processed as nucleotides were retained. described above. Usually, there was no significant lowering of To determine the elution volume and yield of recovery, cyclic the binding when the sample was treated with succinic acid [3H]CMP was added to a biological extract (from urine or blood) (less then 5%); however, when this was not the case, the value prepared in the same way and run in parallel with the samples was regarded as blank and was subtracted from the one in which cyclic CMP was being measured. Over 90% of the obtained with succinylation. Cyclic AMP was determined in the labeled cyclic CMP was recovered. The elution was not moni same sample at the same dilution to confirm its effective tored by addition of cyclic [3H]CMP to each sample because elimination by the purification step. the amount of labeled cyclic CMP would be much higher than Radioimmunoassays of Cyclic GMP and Cyclic AMP. The the content of endogenous cyclic CMP. initial neutral supernatant was also used to determine cyclic When cyclic CMP concentrations were to be determined, the AMP and cyclic GMP by radioimmunoassay (6, 9).

AUGUST 1981 3223

Table 1 Characteristics of antisera against cyclic CMP Rabbit antisera were developed against SCCMP3as immunogen; [125l]iodosuccinylcyclic CMP tyrosine methyl ester was used as labeled antigen. Antisera were diluted in order to obtain 60% of maximal binding, and the binding was measured by equilibrium dialysis. A 10-point displacement curve was constructed for each compound. Cross-reactivity was expressed as the ratio of concentrations giving half-maximal displace ment (i.e., 30% of maximal binding). The dissociation constant calculated from Scatchard plots was given for SCCMP. Cross-reactivity

constant(X Antiserum1C4CDissociation10~'Â°M)6.42.4SCCMP SCAMP1SCUMP CMP1,25011055CMPx 3001 125 10eX

32 160,000SCGMP2,200158,000Cyclic 107 SCCMP; 2'-O-succinyl cyclic cytidine 3':5'-monophosphate; SCUMP, 2'-O-succinyl cyclic uridine 3': 5'-monophosphate; SCAMP, 2'-O-succinyl cyclic adenosine 3':5'-monophosphate; SCGMP, 2'-O-succinyl cyclic guanosine 3':5'-monophosphate.

Table 2 Main clinical and hematological data shown by patients at time of examination Bone marrow Blood

103/cumm)54.38Blasts Patient11907NamecodeC.DeAge20SexFDiagnosis3ALLALLStageRei"ReiBlasts(%)Richness028 (%)0Treatment*1_+ +77 + + + + + +WBC(X 6 mos. later 12738e M.He 81 M ALL A 90 320 98 12957e P.Eu 55 M ALL A 54 5 1 12568e M.An 21 F ALL A 100 94 100 12596 T.Ro 40 M ALL A 88 12.50 44 12597 V.Ja 37 M ALL A 90 14.50 58 ALL Rei 80 11.10 38 6 mos. later

12580 B.Eu 63 M AML A 55 6.30 62 11286 C.Fr 29 M AML Rei 13 9.30 0 For 2 yr

12735 E.Ma 44 AML 81 2.50 36 +

12981eA12818e M.Je 72 M AML 4496 10.804- A13055e H.Ma 19 F AMONOL 4494 440044 A10704e M.Ni 28 F AMONOL 4497' 42.704 498' +76'99'70,919' CLL12894e B.Pa 65 M 4482' 1891504 CLL12363e L.Eu 77 M 4489 CLL12312e C.Lu 73 M 441 40010.609.60â€¢4 CML13007e M.Au 47 M 481 CML12617 P.An 68 M 440 ,9 6'4-63,9 440 34'40 CML12536 B.Mi 70 F 44442004- 4.604 CML12509e R.Yv 70 F 296.5010.103.20-, 462,927' ST11097s M.Ro 71 M +84.912' ST11100s S.Ma 62 M +60,9 STa D.Ge 51 F 28' 4- leukemias."Pathological classification of chemotherapy.c+, with chemotherapy; â€”,without hypercellular;d+ + + + , very hypercellular; + + , moderately + 4, hypercellular;â€¢+normocellular.629794 tumor.9Rei. relapse; A. acute; ST. solid Bloodsampling.' lymphocytes.9Percentage of Percentage of granulocytes.70

In order to assure that succinylation was complete even with Patients the large amount of acylable molecules present in the super natant, the samples were succinylated either undiluted or up to All patients were adults from the Clinique des Maladies du 10-fold diluted and assayed at the same final dilution. Concen Sang (Professor Carcassonne) with complete hematological trations of cyclic AMP and cyclic GMP were measured. No records and their kidney function checked at the time of significant difference was observed between the assays, indi investigation (Table 2). Except when indicated, the patients cating that the succinylation was complete even in undiluted were not under treatment. Six of them had ALL, and 6 had AML samples. including 2 AMONOL's.

3224 CANCER RESEARCH VOL. 41

Urine samples were taken on the first and second day of Cyclic GMP excretions which were similar for AML, ALL, hospitalization. For 8 of them (4 ALL and 4 AML), blood was CML, and solid-tumor patients were lower for CLL patients and taken either on the first or the second day. Three patients with lowest in controls. The excretions of cyclic AMP did not greatly CLL and 4 patients with CML were studied. Blood was taken vary from one disease to another. It was lower in controls. from 5 of them (3 CLL and 2 CML) at the same time as urine Cyclic CMP, Cyclic GMP, and Cyclic AMP Levels in Leu was taken. kocytes. Hyperleukocytic patients, having over 10,000 abnor The urine and blood from 3 patients with solid tumors but mal cells/ cu mm were selected in order to estimate the level having normal differentials and normocellular bone marrow of cyclic nucleotides in leukemic cells. without malignancy were analyzed. Regardless of the cell concentration in the blood, about 5 Urines from 10 healthy volunteers, men and women between x 108 cells were systematically used for cyclic CMP purifica the ages of 25 and 40, having normal hematological counts, tion in order to avoid Chromatographie artifacts due to varia were used as controls. Blood (400 ml) was drawn from 2 men tions in the amount of material loaded on the column. (35 and 39 years old) and one woman (33 years old). Control cells consisted of the Ficoll-isolated leukocytes either As an additional control, PHA-stimulated lymphocytes from from healthy volunteers or from patients with solid tumors. 3 different individuals were analyzed. Cells were obtained from However, for obvious reasons, the number of cells provided by surgically removed tonsils, teased, and resuspended (106 the same person was limited (around 5 x 107 cells for volun cells/ml) in Roswell Park Memorial Institute Medium 1640 teers and up to 3 x 10s cells for solid-tumor patients). supplemented with 10% fetal calf serum and antibiotics, in the Table 4 gives the cyclic nucleotide concentrations in leuko presence of PHA (1 jig/ml). Cell proliferation was measured by cytes. The mean cyclic CMP level was 30-fold higher in acute incorporation of [3H]thymidine. After 2 days of culture, 80% of leukemia than in chronic leukemia. It was impossible to detect the cells were lymphoblasts as estimated by cytofluorography. cyclic CMP in WBC from either controls or solid-tumor patients Cells were harvested at this stage. The viability was about or in PHA-stimulated lymphocytes (even with 109 cells). Cyclic 70%. GMP levels in leukemia cells were not different from those measured in control leukocytes or PHA-stimulated lympho RESULTS cytes. Cyclic AMP concentrations were 10 times lower in cells

Urinary Excretions of Cyclic CMP as Compared with Those of Cyclic AMP and Cyclic GMP. Table 3 shows the urinary " c(IP Â»Mr cANP

excretions of cyclic CMP, cyclic GMP, and cyclic AMP. They g15 are expressed per 10~3 mol creatinine. Cyclic AMP and cyclic

0.1541&uOM.IIM1CHInuTCilIil11io121=â€¢= GMP were in the same range of concentration, whereas the 0.70-oao0.300.1O.Titt!niiMI(UTn'I1o20 level of cyclic CMP was about 2 orders of magnitude lower. The urinary concentration of cyclic CMP was 10 times higher in acute leukemia patients than in healthy controls with an intermediate value for chronic leukemia. Values found in solid IITi1inTCU1il1fi tumors were not significantly different from those found in chronic leukemia. Urinary excretions of cyclic GMP and cyclic AMP were similar in patients with leukemia or solid tumors. In control urines, cyclic GMP and cyclic AMP levels were 5 and 3 times lower, respectively, than in urines of patients. The data (Chart 1) according to the cell type of the leukemia clearly show that urinary cyclic CMP was highest in AML and that the mean value measured in ALL was one-half lower. Chart 1. Excretion of cyclic CMP (cCMP), cyclic GMP (cGMP), and cyclic AMP (cAMP) in urine. Urines were immediately acidified by HCIOÂ«(finalconcen About 2-fold differences were found between AML and CML tration, 1 N); then, the acidic supernatant was neutralized by KOH. An aliquot and between ALL and CLL. The value for solid tumor patients was used for cyclic CMP purification and measurements, another was used for cyclic GMP and cyclic AMP determination, and the third one was used for was between those of CML and CLL; value for controls was creatinine determination. Bars, S.E.; n, number of samples; S7",solid tumor; C, the lowest. control.

Table 3 Urinary excretion of cyclic nucleotides Immediately after collection, urines were made 1 Nwith perchloric acid, and concentrations of cyclic nucleotides and creatinine were determined from the acidic supernatant. Cyclic AMP and cyclic GMP concentrations were measured directly while cyclic CMP concentration was measured after a purification step. The excretion is expressed as mol of cyclic nucleotides per mmol of creatinine. Cyclic CMP (1CT'2mol/mmol GMP (10~6 mol/mmol AMP{10~6 mol/mmol creatinine)AcuteUrines creatinine)0.101 creatinine)0.485 leukemia Â±5.04a (21)" Â±0.012 (21) Â±0.091 (21) Chronic leukemia 9.73 Â±1.61 (10) 0.087 Â±0.020 (10) 0.588 Â±0.106 (10) Solid tumors 9.60 Â±1.38 (3) 0.108 Â±0.029 (3) 0.604 Â±0.100 (3) Controls20.39 2.15 Â±0.352 (20)Cyclic 0.025 Â±0.0078(21)Cyclic 0.192 Â±0.0376(10) Mean Â±S.E. ' Numbers in parentheses, number of patients or, for controls, number of healthy volunteers.

AUGUST 1981 3225

Table 4 Cyclic nucleotide concentrations in WBC Cells were purified on Ficoll density gradient. Leukocytes were harvested and counted. Intracellular concentrations of cyclic nucleotides were measured by radioimmunoassay after prior purification for cyclic CMP. mol/10eCyclic(10~15

WBCAcute CMP1 GMP24.44 AMP403 .26 Â±0.40a (8)" leukemias Â±1 1.92 (5) Â± 66.96 (5) Chronic leukemias 0.046 Â±0.015(5) 9.06 Â± 2.77 (5) 508.8 Â± 2.9 (5) Solid tumors Undetectable (3) 79.25 (2) 2768 (2) Controls Undetectable (3) 15.66Â± 1.90(3) 6186 Â±1562(3) PHA-stimulated lymphocytesCyclic Undetectable (3)Concentration15.33Â± 3.38(3)cells)Cyclic 1687 Â± 662 (3) Mean Â±S.E. ' Numbers in parentheses, number of patients or, for controls, number of healthy volunteers.

:CMP 'M which reflects the overall cell metabolism but also WBC which are directly involved in the disease. The 4 classically defined types of leukemia were studied and compared with different S3 controls. Cyclic GMP and cyclic AMP were measured in parallel , â€ž. to distinguish between general variations of the 3 nucleotide concentrations and a particular change of cyclic CMP level. Our first result was the extremely low level of cyclic CMP as compared to those of cyclic GMP and cyclic AMP which are about 1000-fold higher. In urines, we found that the excretion of the 3 cyclic nucleo tides was higher in patients than in healthy volunteers; the excretions of cyclic GMP and cyclic AMP were in the same range whatever the disease, whereas a high excretion of cyclic r if CMP was clearly associated with acute leukemias. These data were obtained from 10-ml samplings of the first urine of the day instead of a 24-hr urine collection. However, the variability Chart 2. Concentrations of cyclic CMP (cCMP). cyclic GMP (cGMP), and cyclic AMP (cAMP) in leukocytes. Blood leukocytes were purified on Ficoll of the cyclic nucleotide levels from day to day was low and density gradient. Cyclic nucleotides were extracted by HCIO4 (final concentration, always inferior to the differences observed between the dis 1 N). Acidic supernatant was neutralized by KOH. An aliquot was used for cyclic AMP and cyclic GMP radioimmunoassays. The remaining fraction was used for eases. Moreover, these differences between the diseases were cyclic CMP purification and assay. ST. solid tumor; C, control; TC, PHA-stimu significant when the results were expressed either as urinary lated tonsil cells; AMLO, acute monoblastic leukemia. excretion or as urinary concentrations indicating slight variation in the diuresis. of patients with acute and chronic leukemia than in control As we hoped, this particular behavior of cyclic CMP was leukocytes. PHA-stimulated lymphocytes had a cyclic AMP greatly enhanced when we considered its level in WBC, in concentration 4 times lower than that of control cells. which one cell type was largely predominant in most patients Leukocytes of patients with solid tumor contained peculiar tested. Cyclic CMP concentration was 30-fold higher in acute concentrations of cyclic GMP and cyclic AMP. Their cyclic than in chronic leukemia, while this nucleotide was undetect- GMP levels were the highest, and their cyclic AMP concentra able in solid-tumor and control leukocytes as well as in PHA- tions were intermediate between those of leukemic patients stimulated lymphocytes. These data collectively suggest that and controls. the high level of cyclic CMP found in blasts from acute leukemia The detailed data for each class of leukemia make clear the is not related to their high rate of proliferation or their blastic specific behavior of cyclic CMP which was considerably higher state but to some other parameter specific to acute leukemic in acute leukemia than in chronic leukemia. As in urine, the cells, since cyclic CMP was undetectable within PHA-stimu cyclic CMP concentration was maximal in leukocytes from AML lated lymphoblasts. One such parameter might be the stage of (Chart 2). the cells (most of leukemic blasts are in G0, whereas PHA The distinction between AML and AMONOL makes it appar lymphoblasts are in S phase). Cyclic AMP concentration was ent that the higher level of cyclic GMP observed in acute 10 times lower in leukemic cells than in control cells and was leukemia was mostly due to the high level found in AMONOL. also low in PHA-stimulated lymphoblasts. These data extend Hence, AMONOL excepted, the cyclic GMP concentration in and confirm earlier observations indicating that the activity of leukemic cells was in the same range as it was in control cells. cyclic AMP phosphodiesterase is increased in leukemic lym Cyclic AMP concentration was low in all types of leukemic cells phocytes (12) and more recent results showing that the cyclic as compared to control and solid-tumor leukocytes. AMP level was very low in acute lymphocytic leukemia lympho blasts as compared to normal lymphocytes (1). DISCUSSION This low cyclic AMP concentration found both in leukemic cells and in PHA-stimulated lymphocytes suggests that the low The aim of this work was to determine whether the cyclic level is related to the high proliferation rate and/or activation CMP could be considered as a biochemical marker for certain which characterize both leukemic cells and PHA-stimulated types of leukemias. Therefore, we investigated not only urine lymphoblasts.

3226 CANCER RESEARCH VOL. 41

Except in leukocytes from monoblastic leukemia and solid- Adv. Cyclic Nucleotide Res., 5. 331-338, 1975. 4. Bloch, A., Dutschman, G., and Maue, R. Cytidine 3',5'-monophosphate. II. tumor patients where it is higher, cyclic GMP concentration did Initiation of leukemia L1210 cell growth in vitro. Biochem. Biophys. Res. not vary significantly between leukemic and normal cells. Commun., 59. 955-959, 1974. 5. Boyum, A. Separation of white blood cells. Nature (Lond.), 204. 793. 1964. From a clinical point of view, 2 of our findings merit further 6. Cailla, H. L., Racine-Weisbuch, M. S., and Delaage, M. A. Adenosine 3',5'- investigations: the low intracellular levels of cyclic AMP asso cyclic monophosphate assay at 10 15mole level. Anal. Biochem.. 56. 394- ciated with leukemic diseases, whatever the cell line affected 407, 1973. 7. Cailla, H. L., Roux, D., Delaage, M. A., and Goridis. C. Radioimmunological and whatever the state of differentiation; and the high levels of identification and measurement of cytidine 3'.5'-monophosphate in rat tis cyclic CMP characteristic of acute leukemias, whether myelo- sues. Biochem. Biophys. Res. Commun., 85. 1503-1509, 1978. 8. Cailla, H. L., Roux, D., Kuntziger, H., and Delaage, M. A. Antibodies against blastic, monoblastic, or lymphoblastic. Cyclic CMP concentra cyclic AMP, cyclic GMP, cyclic CMP. Their use in high performance radioim- tions could be used as an additional parameter for the diagnosis munoassay. In: J. Dumont and E. Nunez (eds.), Hormones and Cell Regu of the type of leukemia, particularly to distinguish between lation, Vol. 4, pp. 1-25. North Holland: Elsevier. 1980. 9. Cailla, H. L.. Vannier, C. T.. and Delaage, M. A. Guanosine 3',5'-cyclic acute and chronic leukemias and to monitor the blastic trans monophosphate assay at 10 '5 mole level. Anal. Biochem., 70. 195-202, formation of CML's. 1976. 10. Cheng, Y-C., and Bloch, A. Demonstration in leukemia L-1210 cells of a phosphodiesterase acting on 3':5' cyclic CMP but not on 3':5'-cyclic AMP ACKNOWLEDGMENTS or 3':5'-cyclic GMP. J. Biol. Chem.. 253. 2522-2524, 1978. 11. Hadden, J. W., Hadden, E., and Goldberg, N. D. Cyclic GMP and cyclic The authors are deeply indebted to Drs. F. Kourilsky and C. Mawas for their AMP in lymphocyte metabolism and proliferation. In: W. Braun, L. M. invaluable advice and suggestions and to Dr. M Delaage for his permanent Lichtenstein, and C. W. Parker (eds.), Cyclic AMP, Cell Growth, and the interest. They thank B. Malissen for preparations and culture of PHA-stimulated Immune Response. Proceedings of the Symposium held at Marco Island, lymphocytes. Florida, 1973. p. 237. Berlin: Springer-Verlag, 1974. 12. Hait. W. N., and Weiss, B. Increased cyclic nucleotide phosphodiesterase activity in leukaemic lymphocytes. Nature (Lond.), 259. 321-323, 1976. REFERENCES 13. Kuo, J. F., Brackett. N. L.. Shoji, M., and Tse, J. Cytidine 3':5'-monophosphate phosphodiesterase in mammalian tissues. Occurrence and biological 1. Ben Zvi, A., Russell, A.. Shneyour, A., and Trainin, N. Cyclic AMP in human involvement. J. Biol. Chem., 253. 2518-2521, 1978. lymphocytes: levels in acute leukemia and infectious mononucleosis. Eur. J. 14. Pastan, l. H., Johnson. G. S., and Anderson, W. B. Role of cyclic nucleotides Cancer, 13.615-617, 1979. in growth control. Annu. Rev. Biochem., 44: 491-522, 1975. 2. Bloch, A. Cytidlne 3'.5'-monophosphate. I. Isolation from extracts of leuke 15. Scavennec, J., Blanc. A., Gastaut, J.-A., Carcassonne, Y., and Cailla, H. L. mia L1210 cells. Biochem. Biophys. Res. Commun., 58. 652-659, 1974. Cyclic CMP in leukemia diseases. In: P. Hamet and H. Sands (eds.). 3. Bloch, A. Isolation of cytidine 3',5'-monophosphate from mammalian tissues Advances in Cyclic Nucleotide Research, Vol. 12. p. 417. New York: Raven and body fluids and its effects on leukemia L1210 cell growth in culture. Press. 1979.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1981 American Association for Cancer Research. Relationship between the Levels of Cyclic cytidine 3′:5′ -Monophosphate, Cyclic Guanosine 3′:5′-Monophosphate, and Cyclic Adenosine 3 ′:5′-Monophosphate in Urines and Leukocytes and the Type of Human Leukemias

Joëlle Scavennec, Yves Carcassonne, Jean-Albert Gastaut, et al.

Cancer Res 1981;41:3222-3227.

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