To observe the possible role of cAMP on the DNA
synthesis during specialization-division of myelogenous precursor cells, the authors observed the DNA and RNA synthesis of the cells by in vitro autoradiography. And it is concluded that cAMP or its dibutyryl derivative added to the media penetrated into myelogenous precursor cells and metamyelocytes of mice and enhanced the DNA
synthetic capacity of them. cAMP hardly enhanced RNA synthesis. Discussion is made on relation between enhancement of DNA synthesis of metamyelocytes and their possible rejuvenation.</p

Awai, Michiyasu

Naito, Mitsuko

Okada, Shigeru

Okamoto, Atsuko

Seno, Satimaru

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 28, Issue 2 1974 Article 2APRIL 1974Enhancement of DNA synthesis of mousemyelogenous cells by cyclic adenosine3’,5’-monophosphate (cAMP)Shigeru Okada∗ Atsuko Okamoto† Michiyasu Awai‡Mitsuko Naito∗∗ Satimaru Seno††∗Okayama University,†Okayama University,‡Washington University,∗∗Okayama University,††Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Enhancement of DNA synthesis of mousemyelogenous cells by cyclic adenosine3’,5’-monophosphate (cAMP)∗Shigeru Okada, Atsuko Okamoto, Michiyasu Awai, Mitsuko Naito, and SatimaruSenoAbstractTo observe the possible role of cAMP on the DNA synthesis during specialization-division ofmyelogenous precursor cells, the authors observed the DNA and RNA synthesis of the cells by invitro autoradiography. And it is concluded that cAMP or its dibutyryl derivative added to the mediapenetrated into myelogenous precursor cells and metamyelocytes of mice and enhanced the DNAsynthetic capacity of them. cAMP hardly enhanced RNA synthesis. Discussion is made on relationbetween enhancement of DNA synthesis of metamyelocytes and their possible rejuvenation.∗PMID: 4368682 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 28, 81-87 (1974)ENHANCEMENT OF DNA SYNTHESIS OF MOUSEMYELOGENOUS CELLS BY CYCLIC ADENOSINE3', 5'-MONOPHOSPHATE (cAMP)Shigeru OKADA, Atsuko OKAMOTO, Michiyasu AWAI*,Mitsuko NAITO and Satimaru SENDDepartment of Pathology, Okayama University Medical School,Okayama, Japan (Director: Prof. S. Seno)Received for publication, November 17, 1973Abstract: To observe the possible role of cAMP on the DNAsynthesis during specialization-division of myelogenous precursorcells, the authors observed the DNA and RNA synthesis of the cellsby in vitro autoradiography. And it is concluded that cAMP or itsdibutyryl derivative added to the media penetrated into myelogenousprecursor cells and metamyelocytes of mice and enhanced the DNAsynthetic capacity of them. cAMP hardly enhanced RNA synthesis.Discussion is made on relation between enhancement of DNA syn-thesis of metamyelocytes and their possible rejuvenation.It is now recognized that in the multicellular organisms a number ofcellular processes are controlled by the intracellular cyclic adenosine 3', 5'-monophosphate (cAMP), the level of which changes in response to environ-mental stimulation (1). In case of the hormonal stimulation, the hormonecombines with the receptor on cell surface, a part of the adenylcyclase systemin cell membrane, and releases the catalytic part of the enzyme into the cellwhich inevitably results in the formation of cAMP from ATP. For example,glucagon elevates the level of cAMP inside the hepatic cells, which causesthe enhancement of glycogenolysis by the activation of phosphorylase (l), orpituitary growth hormone again induces the production of cAMP in the thymiclymphocytes as has been revealed by in vitro experiments (2).It is still the matter of debate, however, whether cAMP in nutritionalenvironment penetrates into the cells and if so, whether it acts just as thatproduced in the cells. But it has been demonstrated that the cAMP in themedia mimics the action of glucagon stimulating the glycogenolysis of theliver cells in vitro (1), or of pituitary growth hormone promoting the mito-tic activities of the thymic lymphocytes (2). These facts seem to suggestthat in some cells cAMP given from outside penetrates into the cells and* Present address: Division of Hematology, Department of Medicine, Washington Univer-sity, School of Medicine, St. Louis, Missouri, U. S. A.811Okada et al.: Enhancement of DNA synthesis of mouse myelogenous cells by cyclicProduced by The Berkeley Electronic Press, 197482 S. OKADA, A. OKAMOTO, M. AWAI, M. NAITO and S. SENOinduces a similar effect to that of hormones on the target cells. Concerningthe biological potentialities of growth hormone, its stimulating activity ofDNA synthesis and cell proliferation should not be limited on lymphocytesbut on general somatic cells. Therefore, cAMP, as the second messenger ofgrowth hormone, may directly affect the somatic cells other than lympho-cytes. The growth hormone or cAMP may promote the proliferation of thecells in undifferentiated state or in rejuvenation as seen in lymphocytes, butit is quite uncertain whether it has the same activity on the cells on the wayof differentiation and decay. With this problem in mind we have observedthe effect of cAMP on the DNA and RNA syntheses of myelogenous cells ofmouse bone marrow which are destined to mature and to be discarded, andyet the maturation process proceeds with a few steps of DNA synthesis andcell division. As proposed by some authors, their DNA synthesis and prolife-ration may be stimulated by a leukocytosis-inducing factor (3), apart from thepituitary growth hormone. Both of the activities of these two humoral factorsmay be mediated by cAMP involving in DNA synthesis, and if the regulationof DNA synthesis is largely dependent on the phosphorylation of histone bycAMP (4), the activation of DNA polymerase would ultimately result.In this paper it is shown that cAMP added to the incubation media seemsto penetrate into myelogenous precursor cells and to augument DNA synthesisof them without any recognizable enhancement of RNA synthesis, thoughdibutyryl derivative (db cAMP) stimulates both DNA and RNA syntheses.MATERIALS AND METHODSFresh bone marrow cells were obtained from the femurs of healthytwenty adult ddN mice, male and female, weighing about 20 gm. The animalswere decapitated, femurs were taken and cut at the both ends. The bonemarrow tissues were taken out by injecting Hanks' solution containing hepa·'rin, 4 u/ml, from one end. They were put together, dissociated by pumpinggently with syringe and filtered through nylon mesh, then washed twice withHanks' solution by repeated centrifugation I, 500 rpm for S min. and finallysuspended in McCoy SA medium containing 20% calf serum, 106 cells per ml.The cell suspension was divided into 28 parts, one ml each. The first 7 partswere added with cAMP dissolved in saline, 10-2 , 10-3, 10-4 , 10- 5 , 10- 6 , 10- 7 ,and 1O- 8M, respectively and incubated for 3 h at 37°C. The second 7 partsand the third 7 parts were added with db-cAMP and S'AMP respectively atthe same concentration as in the first 7 part and incubated under the same con-ditions. The last 7 parts were of saline control. After 3.h incubation, 3H·Tdrwas added to the incubation medium, 2,uCi,tml (specific activity S Ci/mmole)in each and reincubated for one hour. The reaction was stopped in ice coldbath, washed twice with Hanks' solution by repeated centrifugation and thecells were smeared by cytocentrifugation (6).2Acta Medica Okayama, Vol. 28 [1974], Iss. 2, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss2/2DNA Synthesis by Cyclic AMP 83Autoradiogram was made with these smeared cells according to the me·thod reported elsewhere (5). Five hundred myelogenous precursor cells, in-cluding myeloblasts, promyelocytes, myelocytes and metamyelocytes, werecounted on eaoh sample and labeling index was obtained with these cells.For the observation of RNA synthesis, additional 20 ddN mice were used.Their bone marrow cells were treated by the way exactly identical with thatdescribed above, except that 3H·Ur, 10 ,uCi/ml (specific activity 2. 7 Ci/mmole),and cold thymidine, 1O- 8M, were used in place of 3H-Tdr.RESULTSThe viability test of bone marrow cells isolated from the tissue andincubated under the various conditions described proved that the cells retaineda good state as revealed by morphologic observations and eosin exclusion tests,which were made at the terminal of 3-h incubation by using a drop of eachsample.The myelogenous cells incubated with 3H-Tdr, 2 fLCijml for 60 min.smeared and mounted with sensitive emulsion, gave a moderate number ofsilver grains on the nuclei after one week exposure. Morphologic observationon the cells with May-Grlinwald-Giemsa revealed that the younger precursors,myeloblasts and promyelocytes, were well labeled in all series of the experi-ment. There were no recognizable differences in labeling between the cellsof incubated with cAMP and the controls, as far as these younger precursorcells are concerned. But much differences in labeling were found betweenmore matured cells treated with cAMP and controls. Metamyelocytes andsome mature cells having segmented nuclei were quite rarely labeled in bothsaline controls and 5'AMP-treated samples, while a number of metamyelocytesand some mature cells having segmented nuclei were heavily labeled in thosetreated with cAMP and db-cAMP (Photo I, a and b).On these samples the labeling indices were obtained by counting all seriesof precursor cells, myeloblasts to metamyelocytes. To obtain the labelingindex on each cell series, three grains were required as the limiting graincount per labeled cell. In the samples incubated with cAMP, it was foundthat the labeling index increased at the concentration of 10-4 to 1O- 7M incomparison with the controls incubated with saline. At the concentrationof 10- 7 to 1O-6M, the labeling index increased with the increase in cAMPconcentration. No effect was found at lO- sM and the reduced effects athigher concentration than 1O- 3M (Fig. 1).In samples incubated with db cAMP, a similar increase in DNA syn-thesis was found in myelogenous cells. That is, with the increase in db-cAMPconcentration, 10- 5 to 10- 6M, the labeling index of myelogenous cells in-creased and reached the maximum activation at 1O-4M db-cAMP, 150% of3Okada et al.: Enhancement of DNA synthesis of mouse myelogenous cells by cyclicProduced by The Berkeley Electronic Press, 197484 S. OKADA, A. OKAMOTO. M. AWAI, M. NAITO and S. SENOPhoto 1. Picture of mouse bone marrow cells labeled with 3H-Tdr. Metamyelocytesare labeled rather heavily. The cells were preincubated with cAMP(a) or db-cAMP(b)160r908010/, ....0- ........ "/ "I \/ ~I \I \I \IIIII10-' ,b-' 10-) 10-'Conc~. ,t rat ion (M)Fig. 1. Labeling indices of myelogenous cells of normal mouse bone marrow cells in-cubated with 3H-Tdr for 1 hr at 37°C. Solid line and filled circlles : preincubated with cAMPfor 3 hr at 37°C. Broken lines and open circles: preincubated with db-cAMP for three hoursBroken lines with dots and cross: preincubated with S'AMP for 3 hours. Hatched area:range of controlthe control. Further increase in db-cAMP concentration, 10- 3 to IO-2M db-cAMP, the labeling index lowered to the level of controls (Fig. 1).In the samples incubated with 5'AMP, no activation of DNA synthesis4Acta Medica Okayama, Vol. 28 [1974], Iss. 2, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss2/2DNA Synthesis by Cyclic AMP 85was found. Labeling indices were found to be within the control level atevery concentration tested (Fig. 1).By incubating with aH-Ur and cold thymidine the bone marrow cellsshowed an active RNA synthesis, the younger precursors were more heavilylabeled than more matured cells. The labeling indices of the myelogenouscells preincubated with cAMP and 5'AMP were found to be in the range ofcontrol, while indices of those preincubated with db-cAMP increased alongwith the increase in the concentration of db-cAMP and reached the maximumlevel at 10- 5M, 145% of the control. A further increase in db-cAMP con-centration lowered the index to the control level (Fig. 2)150/;'0-_ .........../ .,/ "',,0I 'I "I ,I 'I "l \I ,/ \~~ \8010 3 1cr' 10-7 lcr'Concentration(M)Fig. 2. Labeling indices of myelogenous cells of normal mouse bone marrow cells in-cubated with 3H-Ur and cold thymidine for one hour at 37°C. Explanation of lines, seeFig. 1DISCUSSIONNow many data are being accumulated on cAMP, which is generallyacccepted as the second intracellular messenger of hormones in multicellularorganism (1). As just described, it is known that cAMP promotes the synthesesof DNA (7, 8, 20), RNA (10, 17, 15, 16) and protein (12, 19) in some cells,e.g. lymphocytes, hepatic cells, etc., but it is still unsettled whether the actionof cAMP, which seems to penetrate some cells from outside and has the pro-moting effect on DNA, RNA and protein syntheses, is specific to some cellsor common to general somatic cells. The present experimental results indicatethat cAMP seems to penetrate mouse myelogenous cell in vitro and to promotethe DNA synthesis. This has been clearly observed on metamyelocytes whichhad a large number of grains after incubation with cAMP and 3H·Tdr, whilethose of controls incubated with saline and 3H-Tdr were poorly labeled. Inthese cells the activation of DNA synthesis by cAMP was induced without5Okada et al.: Enhancement of DNA synthesis of mouse myelogenous cells by cyclicProduced by The Berkeley Electronic Press, 197486 S. OKADA, A. OKAMOTO. M. AWAI, M. NAITO and S. SENOany enhancement of the net synthesis of RNA. It is generally believed thatDNA synthesis requires preliminary synthesis of some RNA and protein (14).The present data showed that cAMP acted to promote the DNA replication butits promoting effect on RNA transcription was very poor in mouse myelogenouscells. This means that cAMP acts mainly to activate DNA replication andrelated RNA synthesis, but poor in the activity of stimulating RNA synthesisfor functioning or structural proteins. The synthesis of RNA for functioningprotein may be related to the factors other than those mediated by cAMP.In this experiment it should be noted that the stimulation of DNA syn-thesis of cAMP and its derivative involves the ring and segmented nuclei ofmetamyelocytes which are generally accepted as fully matured cells havingno mitotic activity. As DNA synthesis occurs actually in these cells, they mayretain mitotic activity which responds to specific stimulation. FANG, HIMEland SENO have reported the possibility of rejuvenation of mature granulocyteor metamyelocyte of rat grafted to the aplastic bone marrow of rat (11, 16).In their observations RNA synthesis preceded DNA synthesis which seemedto occur only after morphologic changes or swelling of nuclei. We have yetno data to explain this discrepancy but DNA stimulating activity of cAMPand derivative on mature granulocyte may give a clue to observe possiblededifferentiation processes of granulocyte as in lymphocyte rejuvenation byPHA.REFERENCES1. SUTHERLAND, E. W.: Cyclic AMP and hormone action. In Cyclic AMP, ed. Robison, G. A.,Butcher, R. W., and Sutherland, E. W., Academic Press, New York and London, 19712. MACMANUS, J. P. and WHITFIELD, J. F.: Mediation of the mitogenic action of growthhormone by adenosine 3', 5'-monophosphate (cyclic AMP). Proc. Soc. Exp. Bioi. Med. 132,409-412, 19693. METCALF, D. and MOORE, M. A. S.: Humoral regulation of haemopoiesis. In HaemopoieticCells, pp.362, North-Holland Pub. Comp., Amsterdam-London, 19714. LANGAN, T. A.: Action of adenosine 3', 5'-monophosphate-dependent histone kinase in vivo.]. Bioi. Chern. 244, 5763-5765, 19695. OKADA, S.: Suppression of erythropoiesis by actinomycin D. II. The change in RNAmetabolism by actinomycin D administration. Acta Med. Okayama 23, 265-271, 19696. DORE, C. F., and BALVOUR, B. M.: A device for preparing cell spreads. Immunology 9, 403-405, 19657. MACMANUS, J. P. and WHITFIELD, J. F.: Stimulation of DNA synthesis and mitotic acti-vity of thymic lymphocytes by cyclic adenosine 3', 5'-monophosphate. Exp. Cell Res. 58,188-191, 19708. WHITFIELD, J. F., MACMANUS, J. P. and RIXON, R. H.: The possible mediation by cyclicAMP of parathyroid hormone-induced stimulation of mitotic activity and deoxyribo-nucleic acid synthesis in rat thymic lymphocytes. ]. Cell Physiol. 75, 213-224, 19709. DELANGE, R.]., KEMP, R. G., RILEY, W. D., COOPER, R. A. and KREBS, E. G.: Activationof skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3', 5'-monophosphate. ]. Bioi. Chem. 243, 2200-2208, 19686Acta Medica Okayama, Vol. 28 [1974], Iss. 2, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol28/iss2/2DNA Synthesis by Cyclic AMP 8710. AVERNER, M. J., BROCK, M. L. and JOST, JP: Stimulation of ribonucleic acid synthesis inhorse lymphocytes by exogenous cyclic adenosine 3', 5'-monophnsphate. ]. BioI. Chern.247, 413-417, 197211. FANG, C. H.: A study of granulopoiesis in the aplastic bone marrow of x-ray irradiatedrats after parabiosis with healthy 1ittermate by aortic anastomoses. Acta Med. Okayarna26, 1-10, 197212. MALLETTE, L. E., EXTON, J. H. and PARK, C.R.: Control of gluconeogenesis from aminoacids in the perfused rat liver. ]. BioI. Chern. 244, 5713-5723, 196913. BURGER, M. and KNYSZYNSKI, A.: The effect of adenosine 3', 5'-monophosphate on 3H·thymidine incorporation in vitro into mouse spleen and thymic cells. Hoppe-Seyler's Z. physiol.Chern. 352, 1019-1024, 197114. MONESI, V.: DNA, RNA, and protein synthesis during the mitotic cell cycle. In Hand-book of Molecular Cytology, ed. A. Lima-De-Faria, pp. 484, North-Holland Pub. Comp.,Amsterdam-London, 196915. DOKAS, L. A. and KLEINSMITH, L. J.: Adenosine 3', 5'-monophosphate increases capacityfor RNA synthesis in rat liver nuclei. Science 172, 1237-1238, 197116. SENO, S., FANG. C. H. and HIMEl, S.: B1astformation of granulocyte: An observation ofhemopoiesis in aplastic bone marrow of x-ray irradiated rat after parabiosis with non-irradiated littermate by aortic anastomosis. (Abstract from XIV International Congressof Haematology, 1972. 7.,17. ARNAUD, M., BEZIAT, Y., BORGNA, J. L., GUILLEUX, J C. and MOUSSERON-CANET, M.: Lerecepteur de l'oestradiol, l'AMP cyclique et 1a RNA polymerase nucleo1aire dans l'uterusde genisse. Stimulation de 1a biosynthese de RNA in vitro. Biochern. Biophys. Acta 25, 241-254, 197118. VARMUS, H. E., PERLMAN, R. L. and PASTAN, 1.: Regulation of lac messenger ribonucleicacid synthesis by cyclic adenosine 3', 5'-monophosphate and glucose. ]. BioI. Chern. 245,2259-2267, 197019. GRIFFIN, D. M. and SZEGO, C. M.: Adenosine 3', 5'·monophosphate stimulation of uterineamino acid uptake in vitro. Life Sciences 7, 1017-1023, 196820. WHITFIELD, J. F., MACMANUS, J. P. and RIXON, R. H.: Cyclic AMP-mediated stimulationof thymocyte proliferation by low concentrations of cortisol. Proc. Soc. Exp. BioI. Med.134, II 70-1 174, 19707Okada et al.: Enhancement of DNA synthesis of mouse myelogenous cells by cyclicProduced by The Berkeley Electronic Press, 1974

Enhancement of DNA synthesis of mouse myelogenous cells by cyclic adenosine 3',5'-monophosphate (cAMP)

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