For the purpose of revealing whether or not hemoglobin synthesis is inhibited by the AMD, the author estimated the hemoglobin level of AMD treated anilmals by microspectrophotometer, and found that the hemoglobin
levels of all the developmental stages of erythroid cells were not inhibited by the AMD. The data indicated that about one half of mRNA for hemoglobin is synthesized in the early stage of specialization with the supplementary synthesis at the later stages and all these mRNA is stable
and insensitive to AMD.</p

Okada, Shigeru

English

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 23, Issue 5 1969 Article 4OCTOBER 1969Suppression of erythropoiesis by actinomycinD. 3. The effect of actinomycin D on thehemoglobin level of bone marrow erythrocytesand erythroblastsShigeru Okada∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Suppression of erythropoiesis by actinomycinD. 3. The effect of actinomycin D on thehemoglobin level of bone marrow erythrocytesand erythroblasts∗Shigeru OkadaAbstractFor the purpose of revealing whether or not hemoglobin synthesis is inhibited by the AMD,the author estimated the hemoglobin level of AMD treated anilmals by microspectrophotometer,and found that the hemoglobin levels of all the developmental stages of erythroid cells were notinhibited by the AMD. The data indicated that about one half of mRNA for hemoglobin is synthe-sized in the early stage of specialization with the supplementary synthesis at the later stages andall these mRNA is stable and insensitive to AMD.∗PMID: 4244047 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. Okayama 23, 387-392 (1969)SUPPRESSION OF ERYTHROPOIESIS BY ACTINOMYCIN DIII. THE EEFECT OF ACTINOMYCIN D ON THE HEMO.GLOBIN LEVEL OF BONE MARROW ERYTHRO·CYTES AND ERYTHROBLASTSShigeru OKADADepartmen! of Pa!hology, Okayama University Medical School, Okayama,Japan (Director: Prof. S. Seno)Received for publication, July 16, 1969In the previous paper it was reported that actinomycin D (AMD) ina small dose of 50 and 100/J.g/kg suppressed the RNA synthesis of earlybasophilic and proerythroblasts without any suppressing effect on theRNA synthesis of erythroblasts in the later stages of specialization asobserved by H3·uridine incorporation and autoradiography (1). This indi-cates that there are at least two kinds of RNA synthesized in erythroblasts ;one mainly in the earlier stages of specialization and sensitive to AMD,and the other mainly in the later stages and insensitive to AMD. TheAMD sensitive RNA synthesized in the early specialization stages will beof rapidly degrading RNA, because in the AMD treated animals theRNA contents of these precursor cells stayed at a certain level withoutshowing any increasing tendency as confirmed by measuring RNA levelwith microspectrophotometer.From the literatures by many authors the rapidly turning-overRNA will be the mRNA and precursors of ribosomal RNA (2), and therole played by the mRNA or mRNAs may be revealed by the proteinproduced under the direction of the mRNA (6). The somatic protein ofthe erythroid cells, hemoglobin, is generally believed to be directed underthe stable mRNA from the observations of reticulocyte (7, 8, 9), and it isthe problem whether the labile, AMD-sensitive RNA is concerned withthe hemoglobin synthesis in the early precursor cells. The absorption ofheme or heme protein at the Soret band appears as early as proerythro.blast stage (10), which is at a very low level, but with the advance ofspecialization stages, it increases, reaching maximum level after denuclea-tion with the active hemoglobin synthesis at reticulocyte stage (11). Thereticulocytes do not synthesize RNA but synthesize hemoglobin actively(7, 11, 12, 13). B~RSOOK et at. reportP-d recently that heme protein otherthan hemoglobin is synthesized in the early specialization stage of erythro.3871Okada: Suppression of erythropoiesis by actinomycin D. 3. The effect ofProduced by The Berkeley Electronic Press, 1969388 S. OKADAblast (14), which will be directed by mRNA specific to this protein,T AKEBAYASHI proved the existence of mRNA for hemoglobin in the veryearly stage of specialization showing high hemoglobin level of macrocytewhich was supposed to be denucleated at an early precursor stage (l0).In referring to those data done by several investigators and theauthor's it was intended to decide whether the AMD sensitive RNA isrelated to the hemoglobin synthesis by observing hemoglobin level percell produced in the animals affected by AMD.In this paper it is reported that AMD sensitive labile RNA is not themRNA for hemoglobin synthesis but probably related to the synthesis ofthe structural protein of erythroblast.MATERIALS AND METHODSSix white adult rabbits of both sexes, weighing about 2kg, were used. Theywere treated with repeated injections of 2.5 % neutralized phenylhydrazine, 0.75to 1mljkg, subcutaneously daily for 3 consecutive days. Three days after thelast injection of phenylhydrazine, 2 animals received a single subcutaneous injec-tion of AMD (Merck-Banyu), 50/-lg/kg and sacrificed 24 hours after AMD admi-nistration. The other two receiving AMD, 50 ltg/kg/day for 3 days, 150llg/kg intotal, were sacrificed 72 hours after the last AMD injection, only proerythro-blasts were observed in the bone marrow smear at this time (19).For the microspectrophotometry of hemoglobin content per cell the bonemarrow smears were made on a cover slide O. 18 x 25 x 50 mm. The bone marrowtissue was obtained from femur, cell suspension was prepared by adding a smallamount of serum and crushing gently in glass homogenizer and smeared. Thesmears were dried and fixed with methanol. Without staining, the measurementsof hemoglobin were taken on the smeared cells by two-wave-length method (3, 4,5) at 406 and 360m/-l, by using Xenon arC (Ushio-UXL-150D) as light sourceattached to the MSP of the Olympus Kogaku Co.RESULTSThe hemoglobin content per cell as observed by microspectrophoto-metric estimation revealed that nucleated cells are extremely low in hemelevel, less than one half of the denucleated cells having the comparablediameter. In erythroblast no increasing tendency of heme level was obser-ved with the advance of cell specialization, but a slightly high level wasobserved in proerythroblast comparing to those of more differentiatedones, No difference was observed in these values between the erythroblastsfrom AMD treated animals and non.treated ones. In erythrocytes includ.ing reticulocytes, the highest value of hemoglobin level was obtained onsome of large cells having diamter of 11 to 12.'\ which were supposed to2Acta Medica Okayama, Vol. 23 [1969], Iss. 5, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss5/4~uppression of Hemopoiesis 389be formed by the denucleation at early basophilic stage. ADd the valuewas 4 times as high as those of the smallest cells having the diameter of 5to 6,1-!, which were supposed to be formed by the denucleation at theorthochromatic stage. In the red cells, having the diameter of 8 to 9,n,the mean hemoglobin level was somewhat lower than that of the largercells, and the level of the cells of 6 to 7,1-! was in between those of the cellsof 8 to 9,1-~ and of 5 to 6,1-!. The data showed no significant differencebetween the two series of experiments, anemic animals treatt.d with AMDand anemic controls without AMD treatment, indicating no effect ofAMD on the heme synthesis.-.: ::-.c-.0::.:.. .• -: \- l. Io _: ..o· --.. ..-.· ..-.: :..· '! ..-... :• •••• 1\ • e.o. ..-0o o...oi.• 0B¥ '0.~ : ..\ 0.. .-.o ... ""a: .JI:;•••• :f.a.~ ..J o~ ~o 0 0 0e8Ci1o°0 c~"" 8o ~0·0o.ooIA00o..23~ 06OL.-,J,.,l--,Jl'=-0--+9----:!8---,7:!:--~6~5Cell diameter (J1)11 10 9 8 7 6 5Cell diameter (,u.)11 10 9 8 7 6 5Cell diameter (,u)Fig. I Relative amount of hemoglobin of erythroblasts, reticulocytes and redblood cells in the bone marrow from the anemic control (A) and anemic rabbitreceiving AMD 50,~~g/kg, sacrificed 24 hours after AMD administration (E) and ofanemic rabbit receiving AMD, 50.Jg/kg/day for 3 days, 150pg/kg in total, sacrificed72 hours after last AMD injection (C). ~'olid circles: red cells and reticulocytes,Open circles: erythroblasts, 5-6fl. orthochromatic erythroblast, 6-8.2,u. polychro-matic erythroblast, 8.2-10.3 fl. late basophilic erythroblast, 10.3-12 fl. early basophilicerythroblast and proerythroblast3Okada: Suppression of erythropoiesis by actinomycin D. 3. The effect ofProduced by The Berkeley Electronic Press, 1969390 S. OI~ADADISCUSSIONPr~s~nt observations revealed that AMD treatment of the anemicanimal did not give any influence on the heme synthesis both of erythro.blasts and red cells; the highest heme level per cell from the anemicanimals of single treatment of AMD and of daily treatment for 3 days,i. e. 3 times AMD dose, were comparable to those from anemic controls.In the anemic control the heme contents of erythroblasts stayed atlower level and rose higher after denucleation, showing that the highestlevel found in large cells of about II to 12/-! was 4 times that of the smallestcells of 5 to 6,n., The data are comparable to those given by T AKEBAYASHIon the anemic rabbits receiving a massive dose of transfusion of red cellsusp~nsion (l0). This and the author's data indicate that about one half ofmRNA for hemoglobin synthesis was synthesized before early basophilicstage, probably at the transformation of the stem cell to proerythroblast,because as just d::scribed, the hemoglobin level seen in the red cellsdenucleated at early basophilic stage was nearly 4 times that seen in cellsdenucleated at orthochromatic stage, and this hemoglobin level is onlyone half of that expected, as it is supposed that the early basophilicerythroblast divides 3 times forming 8 orthochromatic cells, and if mRNAfor hemoglobin were completely synthesized in the early basophilic stage,it would be expected that the hemoglobin level of the nd cells formed bydenucleation at this stage has 8 times that of the normal red cells, 5 to 6 r:,formed by the denucleation at the orthochromatic stage. This indicatesthat mRNA for one half of the total heme expected on those denucleatedat the orthochromatic stage should be synthesized on the way of specializa.tion from proerythroblast to orthochromatic one and it was demonstratedthat actually RNA synthesis insensitive to AMD proceeds all through thestage of specialization of erythroblast (l, II).The present data indicated no appreciable decrease in heme level onthe cells from the animals treated with AMD which inhibited the RNAsynthesis in the early specialization stages of erythroblast, elucidating thatthe AMD sensitive RNA synthesized at the early stage of erythroid cellspecialization is not related to heme synthesis. The author did not observeprotein synthesis but globin synthesis will commence at a very primitivestage of maturation and continues throughout maturation with a lineardecline in synthetic rate (15). It is uncertain to what protein synthesisAMD sensitive RNA is related, but it may be concerned with the synthesisof structural protein as the red cell from the AMD treated animal ~eemsto become smaller in size. Anyway data indicate again that hemoglobin4Acta Medica Okayama, Vol. 23 [1969], Iss. 5, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss5/4~uppression of Hemopoiesisis synthesized by the stable mRNA but not by AMD sensitive one.SUMMARY391For the purpose of revealing whether or not hemoglobin synthesis isinhibited by the AMD, the author estimated the hemoglobin level of AMDtreated anilmals by microspectrophotometer, and found that the hemo-globin levels of all the developmental stages of erythroid cells were notinhibited by the AMD. The data indicated that about one half of mRNAfor hemoglobin is synthesized in the early stage of specialization with thesupplementary synthesis at the later stages and all these mRNA is stableand insensitive to AMD.ACKNOWLEDGMENTThe author is indebted much to Professor SATIMARU SENO for valuable advices throughoutthis work and painstaking proof reading of the paper. Many thanks are also due to MissM. HIRATA for technical assistance and :Cr. M. MIYAHARA for helpful suggestions. Generoussupply of actinomycin D (Dactinomycin) from Japan Merck-Eanyu Co., Ltd. is gratefullyacknowledged.REFERENCESI. OKADA, S.: ~uppression of eryth.opoiesis by actinomycin D. II. The change in RNAmetabolism by actinomycin D administration. Acta Med. Okayama 23, 265, 19692. lINGREL, J. B.: ~tudies on the rapidly labeled RNA of rabbit bone marrow cells, B:ochim.Biophys. Acta 142, 75, 19673. ORNSTEIN, L.; The distributional error in microspectrophtometry. Lab. Invest. 1, 250,19594. PATAU, K.: Absorption microphotometry of irregular shaped object. Chromo:oma 5,341, 19525. MENDELSOHI\', M. L.: The two-wave-Iength method of microspectrophotometry. 1. Amicrospectrophotometer and tests on model system. II. A set of tables to facilitate thecalculations. j. B:ophys. Biochem. Cytol. 4, 407, 19586. JACOB, F. and MO~OD, J.: Genetic regulatory mechanisms in the synthesis of proteins.j. Mol. HoI. 3, 318, 19617. PINHEILO, P., LEBLOND, C. P. and DROZ, B.: ~ynthetic capacity of reticulocytes shownby radioautography after incubation with labeled precursors of protein or RNA. Exp.Cell Res. 31, 517, 19638. MARKS, P. A., BURKA, E. R. and SGHLESSINGER, D.: Protein synthesis in cells. I. Reticu-locyte ribosomes active in stimulating amino acid incorporation. Proc. Nat. Acad. Sc.48, 2163, 19629. MUNRO, A. J. and KORNER, A.: lack of m-RNA in reticulocyte cell-ap. Nature 197,891, 196310. TAKEBAYASHI, J.: Effect of mass blood-transfusion on erythroid cell differentiation inanemic rabbit. II. Denucleation in early stage of erythroid cell specialization, withspecial reference to RNA- and hemoglobin synthesis. Acta Med. Okayama 21, 267, 19675Okada: Suppression of erythropoiesis by actinomycin D. 3. The effect ofProduced by The Berkeley Electronic Press, 1969392 S. OKADA11. SENO, S.: Differentiation of erythroid cell. Acta Path. jap. 16, 457, 196612. SENO, S., ODA, T. and UTSUMI, K.: Hemoglobin synthesis in erythroid cell~. SymposiaCell. Chem. 8, 223, 195813. SENO, S., MIYAHARA, M •• OeHl, 0., MATSUOKA, K., TOYAMA, Y. and SHIBATA, T.:Does reticulocyte synthesize RNA? Acta Med. Okayama 17, 253, 196314. BORSOOK, H., TEIGLER, D. and GUNDERSON, A.: Studies on erythropoiesis. III. Differentpatterns of protein and hemoglobin synthesis before and after terminal differentiationin adult rabbit marrow. Arch. Biochem. BioPhys. 125, 429, 196815. NATHAN, D. G., PIOMELLI, S. and GARDNER, F. H.: The synthesis of heme and globinin the maturing human erythroid cell. j. Clin. Invest. 40, 940, 19616Acta Medica Okayama, Vol. 23 [1969], Iss. 5, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss5/4

Absorption microphotometry of irregular shaped object.

B.: ~tudies on the rapidly labeled RNA of rabbit bone marrow cells,

Differentiation of erythroid cell.

Does reticulocyte synthesize RNA? Acta Med.

Effect of mass blood-transfusion on erythroid cell differentiation in anemic rabbit. II. Denucleation in early stage of erythroid cell specialization, with special reference to RNA- and hemoglobin synthesis.

Genetic regulatory mechanisms in the synthesis of proteins.

Hemoglobin synthesis in erythroid cell~.

lack of m-RNA in reticulocyte cell-ap.

Protein synthesis in cells. I. Reticulocyte ribosomes active in stimulating amino acid incorporation.

S.: ~uppression of eryth.opoiesis by actinomycin D. II. The change in RNA metabolism by actinomycin D administration. Acta Med.

Studies on erythropoiesis. III. Different patterns of protein and hemoglobin synthesis before and after terminal differentiation in adult rabbit marrow.

The distributional error in microspectrophtometry.

The synthesis of heme and globin in the maturing human erythroid cell.

The two-wave-Iength method of microspectrophotometry. 1. A microspectrophotometer and tests on model system. II. A set of tables to facilitate the calculations.

ynthetic capacity of reticulocytes shown by radioautography after incubation with labeled precursors of protein or RNA.

Suppression of erythropoiesis by actinomycin D. 3. The effect of actinomycin D on the hemoglobin level of bone marrow erythrocytes and erythroblasts

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Suppression of erythropoiesis by actinomycin D. 3. The effect of actinomycin D on the hemoglobin level of bone marrow erythrocytes and erythroblasts

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