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

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Relationship Between the Levels of Cyclic Cytidine 3':5'-Monophosphate [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 Joëlle Scavennec,2 Yves Carcassonne, Jean-Albert Gastaut, AndréBlanc, and HélèneL.Cailla3 Centre ¿Immunologie INSERM-CNRS de Marsei/le-Luminy. Case 906, 13288 Marseille cédex9[J. S.. H. L. C.], and Clinique des Maladies du Sang, Hôpital 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 guaríosme 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 anión 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 Thèse de 3e Cycle, University of Aix-Marseille II. Treatment of Samples of Urine and Blood Supported in part by Groupements des Entreprises Françaises 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 —30°.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 20°. 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-Grünwald-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 «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 -80°. 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 4°;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 4°. 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.
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