For the purpose of revealing whether AMD inhibits the RNA synthesis of erythroblasts in an effective dose in vivo to eradicate erythroid cells in rabbit bone marrow, the author observed the RNA synthesis by H3-uridine
incorporation in vitro and RNA level on the cells from the anemic animals taken at a certain period after a single injection of AMD in a small dose of 50 and 100&#956;g/kg body weight.
The data revealed that by such a small dose of injection of AMD the RNA synthesis of erythroid precursors, early basophilic and proerythroblast stages, was successfully suppressed without any suppressing effect on the RNA synthesis of erythroblasts in the later stages of specialization, indicating
that there are at least two kinds of RNA synthesis: one seen mainly in the earlier stages of specialization and the other one seen mainly in the later stages, and they can be distinguished from each other by the AMD sensitivity.</p

Okada, Shigeru

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 23, Issue 4 1969 Article 2AUGUST 1969Suppression of erythropoiesis by actinomycinD. II. The change in RNA metabolism byactinomycin D administrationShigeru Okada∗∗Osaka University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Suppression of erythropoiesis by actinomycinD. II. The change in RNA metabolism byactinomycin D administration∗Shigeru OkadaAbstractFor the purpose of revealing whether AMD inhibits the RNA synthesis of erythroblasts inan effective dose in vivo to eradicate erythroid cells in rabbit bone marrow, the author observedthe RNA synthesis by H3-uridine incorporation in vitro and RNA level on the cells from theanemic animals taken at a certain period after a single injection of AMD in a small dose of 50 and100µg/kg body weight. The data revealed that by such a small dose of injection of AMD the RNAsynthesis of erythroid precursors, early basophilic and proerythroblast stages, was successfullysuppressed without any suppressing effect on the RNA synthesis of erythroblasts in the later stagesof specialization, indicating that there are at least two kinds of RNA synthesis: one seen mainly inthe earlier stages of specialization and the other one seen mainly in the later stages, and they canbe distinguished from each other by the AMD sensitivity.∗PMID: 4243559 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 23, 265-271 (1969)SUPPRESSION OF ERYTHROPOIESIS BY ACTINOMYCIN DII. THE CHANGE IN RNA METABOLISM BYACTINOMYCIN D ADMINISTRATIONShigeru OKADADepartment of Pathology, Okayama University Medical School, Okayama, Japan(Director: Prof. S. Seno)Received fOr publication, May 4, 1969In the previous paper it was reported that the sensitivity of hemopoie.tic organ to actinomycin D (AMD) in vivo administration is considerablyvariable between different species of animal, e. g. mouse and rabbit werehighly sensitive but rat was markedly resistant. In the AMD sensitiveanimals the differentiation of the stem cell to proerythroblast was comple-tely inhibited at a dose of 50,f.tgjkg but the specialization process of theerythroblasts was not suppressed (l). Thus one day after the AMD admini-stration in effective dose, only the erythroblasts in advanced maturationstage were found and after 2 to 3 administrations once a day, the erythro-blasts completely disappeared (1, 2. 3). Such a drastic effect of AMD onhemopoiesis of sensitive animal should be due to the specific affinity ofAMD to erythropoietin, but it may be expected that RNA metabolism isalso affected by the drug, as found originally, AMD binding with DNAmolecule and inhibiting the synthesis of DNA-dependent RNA (4).To settle this problem the author observed the RNA synthesis of theerythroid cells from AMD treated rabbit by flush labeling with HS.uridinein vitro and radioautography. RNA content per cell was also observed bymicrospectrophotomery on these cells on the smear stained with azure B.In this paper it is reported that in the early stage of erythroid cellspecialization, RNA synthesis was suppressed but not in the later stage.RNA level of each cell reflected the suppressed RNA synthesis in the earlystage of cell specialization.MATERIALS AND METHODSEight white adult rabbits of both sexes, weighing about 2kg, were used.They were pretreated with repeated injections of 2.5 % neutralized phenylhydra-zine, 0.75 to 1 ml/kg subcutaneously daily for consective 3 days. Three daysafter the last injection of phenylhydrazine, 4 animals received a single subcuta-neous injection of AMD (Merck-Banyu), 50/log or 100/log/kg. Two of them were2651Okada: Suppression of erythropoiesis by actinomycin D. II. The change inProduced by The Berkeley Electronic Press, 1969266 S. OKADAsacrificed 12 hours after AMD administration, 50,ug/kg. The other two, 24 hoursafter AMD administration, 50,ug/kg. The remaining two also 24 hours afterAMD administration, 100,ug/kg. RNA synthesis by radioautography was obser-ved on the cells from the animals receiving the injection of 50,ug/kg AMD andRNA content per cell was estimated on those from the animals receiving 100,ug/kg AMD injection. Two rabbits served as anemic control.For the observation of RNA synthesis by ridioautography, fresh femur bonemarrow tissue was added with a small amount of the homologous serum and thecells were freed from the tissue by using glass homogenizer, moving gently. Oneml of the cell suspension thus obtained was added with H3-uridine (specific acti-vity 2.7 c/mM, Radiological Centre, England), 10,uc/ml, with cold'thymidine,10 mM/ml. and incubated at 37°C for 1 hour. After incubation the cells werewashed twice with Hanks-serum mixture 0 : 1 vIv) by repeated centrifugationand resuspended in the homologous serum. One drop of the cell suspension wassmeared, fixed with ethanol and dried. After drying the smears were mountedwith nuclear emulsion for radioautography (Sakura NR-M2, diluted with anequal volume of distilled water at 40°C). Then the smears were kept standingvertically in the moist cha.mber (28°C) for 20 minutes and dried at room tempe-rature for 24 hours. The exposure was carried out for one to two weeks in a silicagel chamber at 3°C. After exposure the samples were developed with "KonidolX" at 20°C for 5 minutes, fixed with "Fuji Fix". After fixation these sampleswere washed under tap water for two hours, dipped in a phosphate buffer 0/5M,pH 5.0) at 30 c C for about 15 minutes and stained with Giemsa (Merck). Graincounts were taken of the individual nucleated erythroblasts and two grains wererequired as the limiting grain count per labeled cell. The nuclear diameter andthe grain r.umber were recorded in each cell.RNA l~vels of erythroblasts were estimated on smeared cells by microspectro-photometry. For microspectrophotometry the bone marrow smears were preparedon a cover slide, 0.18 x 25 x 50mm, with the bone marrow cell suspension as justdescribed, and stained with azure B by the slightly modified method of FLAX'and HIMES (5). The smeared cells on cover slide were dried, fixed with methanoland treat.::d with deoxyribonuclease (DNase) solution (crystalized WorthingtonDNase, lmg per ml of Gomori's Tris buffer containing 0.2M MgS04 ·7H20, pH5.7) for ~4 hours at 37°C (6) (on one sample the effect of DNase was tested,reveali:lg negative Feulgen reaction). After DNase treatment the smeared cellswere washed with tap water for 30 minutes, dried, stained with azure B (NationalAniline Division, USA) for 3 hours at 40°C and differentiated in tertiary butylalcohol for 18 hours at 37°C. The azure B was used as 0.025 % solution in Mc-Ilvain's buffer at pH 4.0. On these samples RNA level was estimated by usingthe microspectrophotometer of Olympus Kogoku Co. employing the two-wave-length method deviced by ORNSTEIN (5) and PATAU (6) independently and de-veloped by MENDELSOHN (7) at 590 m/~ and 520 m/~.Identification of the specialization stage of erythroblasts was made by thenuclear diameter according to the principle described by WEICKER (7).2Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/2Suppression of HemopoiesisRESULTS267RNA synthesis of erythroblasts from anemic controls as observed fromthe grain number per cell after in vitro incubation with H3.uridine andradioautography proved to be the highest in the proerythroblast and todecline exponentially with the advance of cell specialization stages, beingminimized at orthochromatic stage. In AMD treated animals the graincount of the cells at more advanced stages were nearly the same as thoseof the cells in the similar specialization stages in anemic control, but inyounger erythroblasts of nuclear diameter of 10 to 13l-t, the grain countsshowed a marked or relatively low levels comparing to those of anemiccontrol. Labeling index was also low in these younger precursors fromAMD treated animal (Fig. 1).The cells having the nuclei of 10 to 13l-t in diameter are of early baso-philic erythroblast or proerythroblast including those in Sand G2 stages.Therefore, the data indicate that the RNA synthesis in early basophilicand proerythroblast stages is AMD sensitive and that in later basophilic,poly. and orthochromatic stages is AMD resistant.RNA content per cell of erythroblasts from anemic control decreasedexponentially in proportion to the decrease in the nuclear volume. But inthose rabbits treated with single injection of AMD 100,ng/kg, the relativeamounts of RNA level per cell of the bone marrow erythroblasts showed amarkedly low level at early stage of specialization comparing to those fromcontrol anemic animal. The RNA level of those at more advanced matura-tion stages showed no distinct difference from those of anemic controls(Fig. 2). The data are consistent with those obtained by observing theRNA synthesis by the incubation with H3.uridine and radioautrographyand indicate that AMD arrests the RNA synthesis of erythroblast only intheir early stage of specialization by in vivo administration in a small doseof 50-100 l-tg/kg body weight.DISCUSSIONIt may be thought that in the erythroid cell specialization AMD actsas to suppress the action of erythropoietin resulting in the inhibition of thetransformation of the stem cell to proerythroblast. But the present obser-vation has clearly demonstrated that AMD suppresses the RNA synthesisin the early stage of specialization of erythroblast in such a small dose of50lf.g/kg irrespective of erythropoietin.It is generally believed that AMD binds with the terminal guanineresidue of DNA and inhibits the synthesis of DNA dependent RNA as has3Okada: Suppression of erythropoiesis by actinomycin D. II. The change inProduced by The Berkeley Electronic Press, 1969s. OKADAB, 3Nuclear diameteqlJ;13 12 IIc-=4 350Fig. 1 The incorporation of H3-uridine into RNA of erythroblasts of anemic rabbitstreated with or without actinomycin D (AMD). The upper figures are the scatter diagramfor grain counts of labeled erythroblasts as classified by the nuclear diameter and thelower ones are labeling indices of erythroblasts at each specialization stage. Pro E: proery-throblast, Baso E. I: early basophilic erythroblast, B. E. II: late basophilic erythro-blast, Poly E: polychromatic erythroblast, Orth E. orthochromatic erythroblastA: anemic controlB: 12 hours after AMD injection, 50 ILg/kgC: 24 hours after AMD injection,50 ILg/kgbeen revealed so far (4, 14). In mammalian cells the similar effect of AMDis expected, but there is a great difference in sensitivity to AMD in thedifferent cell strains. This may be due to the difference in base composi-tion of the DNA chains to be transcribed.Present observations have revealed that the RNA synthesis of erythro-blast of rabbit in the early stage of differentiation is very sensitive toAMD, 50,fI.g/kg, while that in the later stage of specialization is resistantto the same drug in the same dose. This may suggest that the RNA syn-thesized in the earlier differentiation stage may differ from that synthesiz-ed in the later stages, i. e. mRNA synthesized in the earlier stage may bedifferent one from those synthesized later. HARRIS and coworkers, TORELLIand his associates, and LINGREL reported, using AMD as a tool in vitro, of4Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/2Suppression of Hemopoiesis 269A B... ... .... ..·..1098.....::.( ...., .."...... :··.: ....:.......... ...o 19 18 17 16 15 14 13 12 II 10 9 8 7C, Cell dlameter.!I,19 18 17 16 15 14 13 12 II 10 9 8 7Cell diameterl.!/ jFig. 2 Relative amount of azure B binding RNA of erythroblasts and reticulocytesof the bone marrow of the anemic rabbit receiving 100 fLg/kg actinomycin D, sacrificed24 hours after actinomycin D injection (A) and of anemic control (B)7- 8,«: orthochromatic erythrobla~t8-11 fL: polychromatic erythrobla~t11-13fL: late basophilic erythroblast13-18fL: early basophilic erythroblast18-19fL: proerythroblastthe rapidly turningover RNA which appears temporarily in the early stageof erythroid cell specialization and disappears soon (8, 9, 13). This RNAfound by them may be the same one as the AMD.sensitive RNA found bythe author. HARRIS has shown that in cells exposed to AMD the breakdownrate of rapidly labeled nuclear RNA does not proceed in proportion to thedecrease in the protein synthesis. On this finding he has suggested thatmost of the rapidly labeled and short.lived RNA is not functioning asmRNA (8), while AMENTROUT et al. have presented evidence in support ofa relationship between the breakdown of rapidly labeled RNA and proteinsynthesis (10). At any rate the failure in detecting the correlation betweenthe breakdown of rapidly labeled RNA and protein synthesis does not ne-cessarily indicate that this RNA is not mRNA, because the protein direc-ted by this small amount of rapidly turningover RNA might be too small5Okada: Suppression of erythropoiesis by actinomycin D. II. The change inProduced by The Berkeley Electronic Press, 1969270 S. OKADAto be detected by the techniques available at present.On the other hand, the maturation of erythroblasts proceeds withboth cell divisions and synthesis of somatic protein (II). Erythroblasts inthe stages later than early basophilic one, reduce their cell volumes byabout one half at each cell division (7, 11, 12), but it does not necessarilymean the stoppage of protein synthesis. Therefore, the ra pidly turning overmRNA may serve for some structural protein but not the mRNA for he-moglobin, because hemoglobin is synthesized only slightly in the nucleatedstages, especially in early specialization stages (11).Besides these, the specialization of proerythroblast to red cells throughevery maturation steps seems not to be arrested by AMD. During 2 to 3days after AMD injection the recovery curve of anemia is not severelydeviated and no morphological changes of red cells are seen (l). Thesefindings suggest that the rapidly turning over RNA elaborated in earlystage will have no close connection with the cell specialization.In contrast, it is most probable that the mRNA synthsis which appearsin the later stages of cell specialization and proves to be AMD resistantwould be of stable mRNA for hemoglobin, because hemoglobin synthesisbecomes active in later stages, especially after denucleation (II). This isthe problem to be settled in the next step of the author's experiment.SUMMARYFor the purpose of revealing whether AMD inhibits the RNA synthesisof erythroblasts in an effective dose in vivo to eradicate erythroid cells inrabbit bone marrow, the author observed the RNA synthesis by H3.uridineincorporation in vitro and RNA level on the cells from the anemic animalstaken at a certain period after a single injection of AMD in a small doseof 50 and 100rtg/kg body weight.The data revealed that by such a small dose of injection of AMD theRNA synthesis of erythroid precursors, early basophilic and proerythro.blast stages, was successfully suppressed without any suppressing effect onthe RNA synthesis of erythroblasts in the later stages of specialization, in-dicating that there are at least two kinds of RNA synthesis: one seenmainly in the earlier stages of specialization and the other one seen mainlyin the later stages, and they can be distinguished from each other by theAMD sensitivity.ACKNOWLEDGMENTThe author is indebted much to Professor SATIMARU SENO for valuable advices throught6Acta Medica Okayama, Vol. 23 [1969], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss4/2Suppression of Hemopoiesis 271this work and painstaking proof reading of the paper. Many thanks are also dlAe to MissM. HIRATA for technical assistance and Dr. M. MIYAHARA for helpful suggestions. Generoussupply of actinomycin D (Dactinomycin) from Japan Merck.Banyu Co., Ltd. is gratefullyacknowledged.REFERENCESI. OKADA, S.: Inhibition of erythropoiesis by actinomycin D, I. Evolutional change of thebone marrow cells during the drug administration, Acta Med. Okayama 23, 165, 19692. REISSMANN, K. R. and ITO, K. : Selective eradication of erythropoiesis by actinomycinD as the result of interference with hormonally controled effector pathway of cell dif-ferentiation. Blood 28, 201, 19663. SCARO, J. L.: Suppression of erythropoiesis by actinomycin D in normal and erythro-poietin treated mice. Acta Physiol. Lat. Amer. 17, 88, 19674. REICH, E.: Biochemistry of actinomycin. Cancer Res. 23, 1428, 19635. FLAX, M. H. and HIMES, H.: Microspectrophotometric analysis of metachromatic stain-ing of nucleic acids. Physiol. Zool. 25, 207, 19526. AMANO, M.: Improved techniques for the enzymatic extraction of nucleic acids fromtissue sections. j. Histochem. Cytochem. 10, 297, 19627. WEICKER, H.: Zellteilung und ZellteilungsstOrungen, in "Handbuch der gesamten Ha-matologie", 2 Auflage, Band I (ed. L. HEILMEYER and A. HITTMAIR) Munchen, Berlin,Wien, Urban and Schwarzenberg. 1957, pp. 1488. HARRIS, H.: Function of the short-lived ribonucleic acid in the cell nucleus. Nature201, 863, 19649. TORELLI, U., ARTUSI, T., GROSSI, G. EMILIA, G. and MAURI, C.: Unstable ribonucleicacid in normal human erythroblasts. Nature 207, 755, 196510. ARMENTROUT, S. A., SCHMICKEL, R. D. and SIMMONS, L. R.: Actinomycin effect onerythroid messenger ribonucleic acid. Arch. Biochem. Biophys. 112, 304, 196511. SENO, S.: Differentiation of erythroid cell. Acta Path. jap. 16, 457, 196612. WEICKER, H.: Morphologie und Kinetik der normalen und pathologischen Erythropoese.Folia Haemt. N. F. 9, 153, 196413. LINGREL, J. B. : Studies on the rapidly labeled RNA of rabbit bone marrow. Biochem.Biophys. Acta. 142. 75, 196714. REICH, E., CERAMI, A. and WARD, D. C.: Actinomycin, in "Antibiotics" vol. 1, (ed.D. GOTTLIEB and P. D. SHAW) Springer-Verlag, Berlin, Heidelberg, New York, 1967,pp. 71415. ORNSTEIN. L.: The distributional errors in microspectrophotometry. Lab. Invest. 1, 250,195916. PATAU, K.: Absorption microphotometry of irregular shaped object. Chromosoma 5, 341,195217. MENDELSOHN, M. L.: The two-wave.length method of microspectrophotometry I. Amicrospectrophotometer and tests on model system, II. A set of tables to facilitate thecalculations. j. Biophys. Biochem. Cytol. 44, 407, 19587Okada: Suppression of erythropoiesis by actinomycin D. II. The change inProduced by The Berkeley Electronic Press, 1969

Suppression of erythropoiesis by actinomycin D. II. The change in RNA metabolism by actinomycin D administration

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