British Journal of Haemafology, 1975, 29, 593.

Effects of Vincristine on Normal and Stimulated Megakaryocytopoiesis in the Rat SHIRLEY EBBE,D. HOWARD, ELIZABETHPHALEN AND F. STOHLMAN, JR

Tujh University School of Medicine, Boston, Mass. (Received 29 July 1974; acceptedfor publication 19 A t g u s t 1974) SUMMARY. Vincristine was given to rats in which thrombocytopoiesis was either normal or acutcly or chronically stimulated by injections of heterologous antiplatelet serum. A single dose of 0 . 3 mg/kg was given intravenously. The drug produced an early and a delayed megakaryocytopenia suggesting that it was toxic to differentiated megakaryocytes as well as to proliferating stem cells. The results support the hypothesis that vincristine-induced thrombocytosis may be due to homeostatic adjustments which, in turn, arc activated as a result of drug-induced cytotoxicity . The drug vincristine (VC) exerts much ofits cytotoxicity by arresting prolifcrating cells at the metaphase stage of mitosis (Cardinali et al, 1963). This appears to be mediated by dissolution of the mitotic spindle (Malawista et al, 1968), and the drug has been shown to precipitate purified microtubular protein in vitro (Bensch et a!, 1969). Microtubules are present in blood platelets and in megakaryocytic cytoplasm, and incubation of these cells with VC results in dissolution of microtubules aiid development of iiitracellular crystalline inclusion bodies (White, 1968a, b; Bchnke & Pedersen, 1974). From these observations, it might bc cxpected that V C would be more cytotoxic to the thrombocytopoietic cell system than to other cells in which microtubular structures may not be an important part of the cellular architecturc. However, the converse has been found to be true. Clinical experience with V C showed that platelet counts frequently increased out of proportion to tlic therapeutic effect of the drug on the malignant diseasc for which it was given (Carbone et al, 1963 ; Robertson & McCarthy, 1969). Both VC and the related drug viiiblastine (VLB) have been reported to produce less hypoplasia of the megakaryocytic cell system than of myeloid and erythroid cells in experimental animals (Boggs et al, 1964; Morsc & Stohlman, 1966). In rats and mice, V C has been reported to result iu thrombocytosis that appeared to be due to stimulation of platelet production by the drug. The platelet response was dose-dependent, but the development of thronibocytosis did not seem to be a secondary phenomenon that was dependent upon a primary reduction in platclets by thc drug in the reports by Robertson et a1 (1970, 1972) and Rak (1972). Klener et al (1972) also found what appeared to be a direct stimulatory effect of VLB on megakaryocytopoiesis and platelet production. Krizsa et ul (1973) came to the somewhat different conclusion that thc thrombocytosis produced by V C in mice was mediated by throinbopoietin produccd in response to Correspondence: Dr Shirley Ebbe, St Elizabeth’s Hospital, 736 Cambridge Street, Brighton, Massachusetts02135 , U.S.A.

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drug-induced thronibocytopenia. Choi et ul(1974) analysed the sequential response of the rat thrombocytopoietic system to a single injection of different doses of VC. Rather than the drug producing thrombocytosis by direct stimulation of the megakaryocytic system, their results indicated that the initial event was depopulation of early megakaryocytes by VC, but not of sufficient degree to result in thrombocytopeiiia when the dosc of VC was small. Secondarily, there appeared to be a compensatory increase in differentiation of megakaryocytes and a shortening of maturation time which tended to ovcrcompeiisate and result in mild thrombocytosis. The megakaryocytic cell system is unique among the haematopoietic cells in that the most of its proliferative activity occurs in morphologically unrecognizable precursor compartments. Cytotoxic agents and homeostatic regulators affect this sytem largely at the precursor cell levels, but some of their effects may be seen from subsequent changes in recognizable megakaryocytes and circulating platelets. The studies to be reported were undertaken to utilize the stathmokinetic effect of VC as a probe to analyse the behaviour of normal and stimulated megakaryocyte precursors by arresting mitosis of diploid cells and also, perhaps, endomitosis of polyploid cells. They also were aimed at further evaluation of the curious finding that VC may actually stimulate platelet production. A single dose of 0.3 mg/kg body weight of VC was used; it was selected because the published experience of others at the time the experiments were begun indicated that it should produce thrombocytosis without antecedent thrombocytopenia. MATERIALS AND METHODS Experiments were done in female Sprague-Dawley rats. Vincristine was given as a single intravenous injection through a tail vein in a dose of 0.3 nigikg. Blood samples were obtained by cardiac puncture under ether anaesthesia following which the rats were killed with ether. Platelet counts were done by phasc microscopy (Brecher & Cronkite, 1950). Tibia2 cell counts. The tibiae were removed, the proximal articular surface shaved off with a razor blade, and the distal portion below the fibular junction was cut off and discarded. Marrow contents were flushed out with z ml of 1% Na,EDTA w/v in isotonic saline and cells were dispersed by repeatedly washing the suspension through the bone. Total nucleated cell count was determined by Coulter counter after dilution in Isoton (Coulter) and saponin. An aliquot of the tibia1 suspension was stained by adding 0.1 volume of new methylene blue. Then megakaryocytes in 0.9 pl were counted by light microscopy. Megukuryocyte sizing. Sizes of individual megakaryocytes were estimated from bone marrow smears as previously described (Ebbe et ul, 1968). Briefly, cells were photographed and their outlines were traced onto paper of uniform weight from projections of the film negatives. The tracings were cut out and weighed to give measures of cell size. Megakaryocytes were classified as stages I, I1 and I11 (successive phases of maturation) as previously described (Ebbe & Stohlman, 1965). Antiserum. Antiserum to rat or mouse platelets was prepared in rabbits and absorbed with rat or mouse red cells as previously described (Ebbe et al, 1968). To produce thronibocytopenia, appropriate dilutions of the antiplatelet serum (APS) were injected iiitraperitoneally in doses of 0.2 m l in rats and 0.1ml in mice.

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Irradiation of mice. CF,, mice (Carworth Farms) were exposed to total body, sublethal, radiation with a caesium source (Gammacell 40, Atomic Energy of Canada Ltd). RESULTS

Efccts of VC in Normal Rats Platelet couiits following administration of V C to normal rats are shown in Fig I. They decreased gradually for the first 2 days, and the nadir was about 70% of normal. Between days

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FIGI . Platelet counts in normal rats after administration ofVC. Each expcrimcntal value is the average (_+SEM) for 5-14 rats; the control (time zero) is the average for 41 rats. FIG 2. Numbers of megakaryocytes ( 0 ) and total nucleated cells (0)per tibia in normal rats after administration of VC. Each experimental value is the average ( & SEM) for 6-14 rats; the control (time zero) is the average for 27 rats.

and 5 , platelet coLiiits increased to reach levels that were modestly thrombocytotic (120% of normal). Thereafter they returned to normal. This sequerice of changes in circulating platelets suggested the presence of a biphasic response to VC, the first part due to the drug itself, and the second perhaps due to compensatory homeostatic adjustments that may have been activated by the initial effects of the drug. Four hours after injection of V C there was a drop of about 40% in the number of megakaryocytcs that were countable in tibia1 marrow cell suspensions (Fig 2). The iiumber then gradually increased to reach normal values on day 2. Subsequently they again fell to about 60% of normal on days 3 and 4.Differential counts of megakaryocytcs by maturation stage on 250 megakaryocytes from each of four rats 3 11, I day and 2 days after administration of VC were normal, thus confirming the findings of Morse & Stolilman (1966). Therefore, it appeared that V C was equally toxic to all cells iii the compartment of recognizable megakaryocytes. Seven mitotic figures were sew iii 340 stage I megakaryocytes analysed during 2

Shirley Ebbe et a1

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the first 12 h after VC was given. The mitotic index (2.0% of stage I or 0.4% of all megakaryocytes) did not change in successive marrows examined at I , 4, 8 and 12 h. This value was somewhat smaller than that reported by Ode11 et al (1968) and Choi et a1 (1974). Total nucleated cells decreased to about half of normal during the first day and remained so for 3 days (Fig 2). After day 4, megakaryocytes and other nucleated cclls increased in parallel to reach normal values by day 6. During the first day after administration of VC, the sizes of individual megakaryocytes remained normal. After that there was a gradual increase in cell size of about 20-40% (Table I, Fig 3) with a return to normal by day 6. Measurements of size for the three stages

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t Each experimental value is the average of 66-74 megakaryocytesfrom two rats. of maturation were made for the first 3 days after VC was given, and it was found that the size change occurred simultaneously in all (Table I).

Efects of VC on Acutely StinlMlated Thrombocytopoiesis When rats were permitted to recover from the thrombocytopenia induced by two daily injections of rabbit antirat platelet serum (APS), their platelet counts gradually increased to thrombocytotic levels (Fig 4).When VC was given after the second dose of APS, recovery of the platelet count was delayed, and thrombocytosis did not develop during the 6 days of observation. VC did not interfere with the development of macromegakaryocytosis in these animals, but rather appeared to prolong its duration (Fig 3). Numbers of tibia1 megakaryocytes were not different from controls for the first 2 days after VC, but they decreased on the third and fourth day (Fig 5 ) . Minimum values were about 25-30% of the number of megakaryocytes in stimulated or control rats that were not given VC. Total number of marrow nucleated cells respondcd to the drug in much the same manner as in unstimulated rats, and recovery of both cell systems was complete by day 6. For comparison, platelet counts were determined in mice subjected to the same degree of thrombocytopoieic stimulation by two daily injections of rabbit antimouse platelet serum; after the second injection, sublethal total body irradiation was substituted for VC. From the results shown in Fig 6, it is clear that recovery from the thrombocytopenia was not impaired as it had been after VC. The subsequent radiation-induced thrombocytopenia was not modified by the immune thrombocytopenia which had been present for 24 h at the time of irradiation.

Effect of Vincristine on Megakaryocytopoiesis

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Efect of VC on Chronically Stimulated Tkronibocytopoiesis More prolonged stimulation of megakaryocytopoiesis was achieved by maintaining thrombocytopenia with four daily injections of APS. Recovery and development ofthrombocytosis occurred more promptly after cessation of APS injections than in the previous group of rats suggesting that, with the longer duration of thrombocytopenia, sufficient time had vc

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FIG 3 . Relative size of stage 111megakaryocytes in rats treated with VC only (A), two daily injections of APS (o), or two daily injections of APS followed by VC ( 0 ) .The average control value was determined for 297 megakaryocytes from eight rats; other points represent the mean (f SEM) for 104-189 megakaryocytes from three to five rats. FIG 4. Platelet counts of rats after two daily injections of APS (on days - I and 0). Open circles represent rats given APS only; rats depicted by closed circles received VC on day o, after the second dose of APS. Each point is the average for three to fivc rats; SEM is shown where the average values appeared to differ.

elapsed for the compensatory changes characteristic of stimulated megakaryocytopoiesis to beconic well established within thc compartment of recognizablc incgakaryocytes. Administration of V C I day after the final APS injection did not alter thc dcvelopnicnt of rcbound thrombocytosis (Fig 7). Thus, thc platclct counts did not give cvidciicc of an early or a dclayed toxic effect of V C 011 megakaryocytopoicsis. Thc tlirombocytosis was also not potentiated by VC, so there was no reason to think that V C had supcriinposed a druginduced stimulation onto that already present.

Shirley Ebbe et a1

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FIG 5. Numbers of tibial megakaryocytes in rats treated with two daily injections of APS (0)or with two daily injections of APS followed by VC ( 0 ) .Controls (day - I) are the average (rf: SEM) for eight rats; other points are the average for three to five rats. FIG 6. Platelet counts of mice exposed to 400 R total body irradiation on day 0.Closed circles represent mice pretreated with APS on days - I and 0. Open circles represent mice that were not pretreated. Each point is the average (+_SEM) for five or six mice; P values (Student's t test) are shown for points that had a 5% or greater possibility of being statistically different.

Average numbers of tibial megakaryocytes were about z0-30% higher than normal after 4 days of APS administration (Fig 8). Their numbers stayed about the same during the succeeding 3 days in rats that did not receive VC and then they began to fluctuate downwards. Changes in megakaryocyte numbers after VC was given followed the same pattern as it had in non-stimulated normal rats given VC. Total marrow nucleated cell numbers were normal after chronic thrombocytopoietic stimulation, and they responded normally to VC.

DISCUSSION In the present experiments,vincristine had a biphasic effecton the number of megakaryocytes in normal rats. Within 4 h of its administration, megakaryocytes were reduced by about 40% ; because of its early appearance, this depletion appeared to be due to direct toxicity to recog-

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FIG 7. Platelet counts of rats in which thrombocytopenia was maintained for 4 days with daily injections of APS (on days -4, - 3 , - 2 and -I). Opcn circles represent rats treated with APS only. Closed circles represent rats in which VC was given on day 0.Each point is the average (f SEM) for four to six rats. FIG 8. Numbers oftibial megakaryocytes in rats treated with four daily injections of APS (0)or APS plus VC ( 0 ) . The control value (day -4) is the average (fSEM) for 13 rats; other points are the average for four to seven rats.

nizable cells. The subsequent decrease in circulating platelets suggested that the early megakaryocytopenia was of physiological significance. The possibility that the thrombocytopenia was due to toxicity of the drug to circulating platelets with shortening of their survival time cannot be ruled out. However, the gradual, rather than precipitous, drop in platelets and the temporal relationship to the early megakaryocytopenia suggests that the thrombocytopenia resulted from a period of reduced platelet production. Gradual recovery of megakaryocytes to normal numbers during the first and second days after VC was presumed to be due to influx of cells from precursor compartments that were not heavily damaged by VC. Examination during this time, when total cellularity a i d megakaryocytes were not proportionately reduced, could lead to the conclusion that niegakaryocytes were relatively unaffected by VC (Morse & Stohlman, 1966). On the third and fourth days after VC, megakaryocytes were reduccd by about the same proportion as total cells. The delayed effect was probably due to an action of VC on early precursor cells that were temporally removed from the Compartment of recognizable megakaryocytes by a maturation time of between z and 3 days. Ode11 et al(1966) found that repeated injections of VLB produced megakaryocytic hypoplasia but only after a delay of z days, indicating toxicity to the same cell population. Morse & Stohlman (1966) found that the erythroid compartment

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Shirley Ebbe et a1

of normal rat marrow was almost totally depopulated by VC given in exactly the same way as was done in our experiments. The maximum dcpletion of megakaryocytes that we found was about 40%. Therefore, these new results are not iiiconsistent with the conclusions of Morse & Stohlman (1966) that megakaryocytopoiesis may be ‘relatively unaffected’ by VC and that there are different immediate precursor cells for the differentiated erythroid and megakaryocytic compartments. The results of Choi et al(1974) suggested that VC may initially damage the megakaryocytic system and that this damage, even though it may not result in peripheral thrombocytopenia when the dose of VC is small (0.1mgikg), may activate compensatory mechanisms. Like the compensatory mechanism activated by peripheral thrombocytopenia, there appeared to be an over-response which resulted in thrombocytosis. Our results with a larger dose of VC are not so clear-cut because of the transient development of thrombocytopenia which, in itself, probably stimulated the megakaryocytic system. During recovery from early megakaryocytopenia, megakaryocyte size increased. The mechanism of this macrocytosis was not clear, but it is possible that it may have represented a manifestation of an intramedullary compensatory mechanism in response to diminished numbers of megakaryocytes,much as macromegakaryocytosis occurs in response to thrombocytopeiiia (Ebbe et al, 1968). Its duration was probably prolonged by the peripheral thrombocytopenia but its onset seemed to be too early to be attributable to peripheral thrombocytopenia alone. The changes observed during the first 2 days after VC tended to support the conclusions of Choi et al(1974) and to show the possible importance of homeostatic mechanisms within hematopoietic tissues which may not be dependent on changes in numbers of circulating cells. Recovery from thrombocytopeiiia and development of modest thrombocytosis in the presence of the secondary reduction in numbers of megakaryocytes may have been accomplished, in part, by the macromegakaryocytosis and, in part, by the acceleration of megakaryocyte maturation that Choi et al(1g74) observed. It has been considered (Ebbe, 1971) that megakaryocytopoiesis consists of concatenate cellular compartments in the following sequence :

(A) (B) (C) (D)

Pluripotential stem cells. ‘Resting’ committed stem cells. Committed stem cells undergoing cellular proliferation (2N megakaryocytes). Precursor cells undergoing nuclear replication (including about half of the cells identifiable as immature, stage I, megakaryocytes) (Odell et a / , 1968). (E) Recognizable non-proliferating megakaryocytes. Analysis of our results suggests that VC is toxic to cells in compartments (C) and (E). Regeneration of numbers of recognizable megakaryocytes for 2 days after VC suggests that compartment (D) may not be heavily damaged by the drug. This seems curious in view of the description of mitotic division of megakaryocyte nuclei (Japa, 1943) and the polyploid metaphase arrest that is induced by incubation in vitro with colchicine (Rolovic, 1974). Individual megakaryocytes have been shown to contain quantities of DNA that approximate 2XN,thus indicating that polyploidy is achieved by successive doubling of nuclear DNA (Odell et al, 1965). If, with each doubling, a mitotic spindle formed and nuclear division occurred, then it would be expected that megakaryocytes would contain multiple nuclei, each with a diploid

Eject of Vincristine on Megakauyocytopoiesis

60I

amount of DNA. O n the contrary, the DNA of megakaryocytes has been shown to be contained in a single nucleus which bccomcs segmented, for the most part, after cessation of D N A synthesis (Paulus, 1970; dcLeval & Paulus, 1971). These considerations and our failure to find an accumulation of stage I megakaryocytes in metaphase during the 12 h after adniinistration of V C suggest that if true nuclear mitosis occurs it is the exception rather than the rule. Thus, the apparent lack of effect of V C on this compartment could be attributed to the fact that spindle formation does not occur. It would be expected from the work of Sinclair (1968) and Cardinali et al(1963) that radiation and V C would affect the same cellular populations since both agents are maximally toxic to cells in mitosis. Other rcsults (Ebbe & Stohlman, 1970; Odell et al, 1971) indicated that the major site of sensitivity to radiation was compartment (C). If radiation and V C both affected the same compartment, then why did VC not produce the delayed thrombocytopenia characteristic of radiation? It is possible that the doses of the two agents were not comparable and that different numbers of cells were, therefore, damaged. Another possibility is that the early VC-induced megakaryocytopeiiia or tlirombocytopenia may have initiated a compensatory increase in platelet production. In response to peripheral thrombocytopenia, several megakaryocytic changes have been described which have been interpreted as responses to stimulation. The simplest explanation for many of the changes is that all processes of cellular maturation, including nuclear replication with developinent of polyploidy (D), are accelerated and that early precursor cells (B) are stimulated to differentiate into the proliferating compartment (C). When V C was given to rats with acutely stimulated thrombocytopoiesis, recovery from immunothrombocytopcnia was delayed, while it was not hindered by radiation. This confirmed the observation that V C damaged cells of the megakaryocytic series which were more mature than those damaged by radiation. Early VC-induced megakaryocytopeiiia was not demonstrable in these animals, but the delayed reduction was more severe than in normals suggesting that a greater proportion of cells in compartment (C) were in cell cycle than normally. Lack of potentiation of radiosensitivity suggests that post-irradiation repair of compartments occurred. V C did not interfere with development of macromegakaryocytosis, thus further indicating that the drug did not damage compartment (D), since megakaryocyte size has been shown to be proportional to nuclear ploidy (Odell et af, 1970). In animals recovering from more chronic stimulation, V C did not interfere with development of rebound thrombocytosis, and, in this respect, its effect was not different from that of sublethal irradiation (Ebbe & Stohlman, 1970). There was no evidence of either suppression or stimulation. Mcgakaryocyte counts followed the same pattern as in normal rats given VC, but, since the starting value was higher, the absolute values wcrc somewhat greater than in uiistimulated normal rats. While there was no evidence that V C effected any substantial reduction in platelet production during recovery from chronic thrombocytopenia, there was also no evidence that platelet production was further stimulated by the drug. 111the chronically stiinulated rats, the early drop in megakaryocyte numbers did not result in values below those normally present, and this may have accounted for the failure of further stimulation to occur. In the chronically stimulated state, conditions may be coniparablc to those present in human beings with prolonged chronic idiopathic thrombocytopeiiic purpura (ITP) of the refractory

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type in which immunosuppressive therapy may be conternplatcd (Aisenberg & Wilkes, 1964; Sultan et a!, 1971 ; Harrington et a/, 1972). Although V C induced changes in megakaryocyte number, the chronically stimulated system was able to compensate for the changes, thus suggesting that appropriately timed injections of the drug would be tolerated well in ITP. The more profound effect on the acutely stimulated system suggests that the drug might be potentially harmful to patients in whom compensatory adjustments of megakaryocytopoiesis were occurring in response to platelet depletion of recent onset such as might occur with druginduced thrombocytopenia, defibrination, or blood replacement. However, it should be noted that the dose of V C used in these studies was about ten times the dose ordinarily given to human beings, so application of the results to human beings must remain conjectural. ACKNOWLEDGMENTS

This work was supported in part by Grant AM-08263 from the National Institute of Arthritis, Metabolism and Digestive Diseases, Grant E T - I ~ Bfrom the American Cancer Society, Inc., and Grant HL-07542 from the National Heart and Lung Institute. REFERENCES AISENBERG, A.C. & WILKES,B. (1964) Studies on the suppression of immune responses by the periwinkle alkaloids vincristine and vinblastine. Journal of Clinical Investigation, 43, 2394. BEHNKE, 0. & PEDERSEN, N.T. (1974) Ultrastructural aspects of megakaryocyte maturation and platelet release. Platelets: Production, Function, Transjusion and Storage (Ed. by M. Baldini and S. Ebbe), p 21. Grune & Stratton, New York. H. & BENSCH,K.G., MARANTZ,R., WISNIEWSKI, SHELANSKI, M. (1969) Induction in vitro of microtubular crystals by vinca alkaloids. Science, 165,495. BOGGS, D.R., ATHENS, J.W., HAAB,O.P., CANCILLA, P.A., RAAB,S.O., CARTWRIGHT, G.E. & WINTROBE, M.M. (1964) Leukokinetic studies. VII. Morphology of the bone marrow and blood of dogs given vinblastine sulfate. Blood, 23, 53. BRECHER, G. & CRONKITE, E.P. (1950) Morphology and enumeration of human blood platelets. Journal of Applied Physiology, 3, 365. CARBONE, P.P., BONO,V., FREI,E., 111, & BRINDLEY, C.O. (1963) Clinical studies with vincristine. Blood, 21,640.

CARDINALI, G., CARDINALI, G. & ENEIN,M.A. (1963) Studies on the antimitotic activity of leurocristine (vincristine). Blood, 21, 102. CHOI, S.I., SIMONE, J.V. & EDWARDS, C.C. (1974) Effects of vincristine on platelet production. Platelets: Production, Function, Transjiision and Storage (Ed. by M. Baldini and S. Ebbe), p 51. Grune & Stratton, New York. DELEVAL, M. & PAULUS, J.M. (1971) Megakaryocytes: Uninucleate polyploid or plurinucleate cells?

Platelet Kinetics (Ed. by J. M. Paulus), p 190. North-Holland Publishing Co., Amsterdam. EBBE,S. (1971) Origin, production, and life span of blood platelets. The Circulating Platelet (Ed. by S. A. Johnson), p 19. Academic Press, New York. EBBE,S. & STOHLMAN, F., JR (1965) Megakaryocytopoiesis in the rat. Blood, 26, 20. EBBE,S. 81 STOHLMAN, F., JR (1970) Stimulation of thrombocytopoiesis in irradiated mice. Blood, 35, 783.

EBBE,S., STOHLMAN, F., JR, OVERCASH, J., DONOVAN, J. & HOWARD,D. (1968) Megakaryocyte size in thrombocytopenic and normal rats. Blood, 32, 383.

HARRINGTON, W.J., AHN,Y .S. & GILIBERTI, J.J. (1972) Vincristine therapy of idiopathic thrombocytopenia. (Abstract). Blood, 40, 971. JAPA,J. (1943) A study of the morphology and development of the megakaryocytes. British Journal of Experimental Pathology, 24, 73. KLENER,P., DONNER,L. & HOUSKOVA, J. (1972) Thrombocytosis in rats induced by vinblastine. Haemostasis, I, 73. KRIZSA,F., KOVACS, Z. & DOBAY,E. (1973) Effect of vincristine on the megakaryocyte system in mice. Journal of Medicine, 4, 12. MALAWISTA, S.E., SATO,H. & BENSCH, K.G. (1968) Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science, 160, 770. MORSE,B.S. & STOHLMAN, F., JR (1966) Regulation of erythropoiesis. XVIII. The effect of vincristine and erythropoietin on bone marrow. Journal of Clinical Investigation, 45, 1241.

Effect of Vincristine on Mega karyocytopo iesis ODELL,T.T., JR,JACKSON, C.W. & FRIDAY, T.J. (1970) Megakaryocytopoiesis in rats with special refcrcncc to polyploidy. Blood, 35, 775. C.W. & FRIDAY, T.J. (1971) ODELL, T.T., JR,JACKSON, Effects of radiation on the thrombocytopoietic system of mice. Radiation Research, 48, 107. D.G. ODELL,T.T., JR, JACKSON, C.W. & GOSSLEE, (1965) Maturation of rat megakaryocytes studied by microspectrophotometric measurement of DNA. Proceedings of the Society for Experimental Biology and Medicine, 119, 1194. ODELL, T.T., JR,JACKSON, C.W. &REITER,R.S. (1968) Generation cycle of rat megakaryocytes. Experimental Cell Research, 53, 321. ODELL,T.T., JR, JEFFERSON, M., JACKSON, C.W. & REITER,R.S. (1966) Effects of vinblastine on megakaryocytes and platelets of rats. Experimental Fernatology No. 10, p 36. Biology Division, Oak Ridge National Laboratory. PAULUS, J.-M. (1970) DNA metabolism and development of organelles in guinea-pig megakaryocytes : a combined ultrastructural, autoradiographic and cytophotometric study. Blood, 35, 298. RAK,K. (1972) Effect of vincristine on platelet production in mice. BritishJournal of Haematology, 22, 617. ROBERTSON, J.H., CROZIER, E.H. & WOODEND, B.E. (1970) The effect of vincristine on the platelet count in rats. British Journal ofHaernatology, 19, 331.

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ROBERTSON, J.H., CROZIER, E.H. & WOODEND, B.E. (1972) Vincristine-induced thrombocytosis studied with 75% selenomethionine. Acta Haernatologica, 47, 356. ROBERTSON, J.H. & MCCARTHY, G.M. (1969) Periwinkle alkaloids and the platelet-count. Lancet, ii, 353.

ROLOVIC, Z . (1974) Ploidy value of endoreduplicating human megakaryocytes in immune and ‘hypersplenic’ thronibocytopenia. Platelets: Production, Function, Trandusion and Storage (Ed. by M. Baldini and S. Ebbe), p 143. Grune & Stratton, New York. SINCLAIR, W.K. (1968) Cyclic X-ray responses in mammalian cells in oitro. Radiation Research, 33, 620.

SULTAN, Y., DELOBEL, J., JEANNEAU, C . & CAEN,J.P. (1971) Effect of periwinkle alkaloids in idiopathic thrombocytopenic purpura. Lancet, i, 496. WHITE,J.G. (1968a) Effects of colchicine and vinca alkaloids on human platelets. I. Influence on platelet microtubules and contractile function. American Journal OfPathology, 53, 281. WHITE,J.G. (1968b) Effects of colchicine and vinca alkaloids on human platelets. 11. Changes in the dcnse tubular system and formation of an unusual inclusion in incubated cells. American Jorirnal o j Pathology, 537 447.