Cell Tissue Kinet. (1976) 9, 363-369.
T H E EFFECTS O F COLCEMID O N M O U S E B O N E MARROW STANLEY E. SHACKNEY, PAUL A. BUNN, JR A N D SHARONs. FORD Ofice of the Director, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland (Received 15 October 1975; revision received 15 January 1976) ABSTRACT
Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes. INTRODUCTION Preferential regional uptake of tritiated thymidine (3H-TdR) has been demonstrated radioautographically in a variety of tissues, including intestinal epithelium (Quastler & Sherman, 1959), epidermis (Leblond, Greulich & Pereira, 1964; Iversen, Bjerknes & Devik, 1968), regenerating liver (Grisham, 1962), thymus (Borum, 1968, 1973), lymphoid germinal centers (Hanna, 1964; Ishii, Mori & Onoe, 1972), adrenal (Diderholm & Hellman, 1960; Ford & Young, 1963; Wright, 1971) and certain tumors (Tannock, 1968). In recent studies in mouse bone marrow it has been shown that the subendosteal region of the marrow exhibits the highest labelling index and the largest number of heavily labelled cells 2 hr after pulse 3H-TdR exposure (Shackney, Ford & Wittig, 1975). Frequently, the areas of greatest 3H-TdR uptake are also those which are endowed with the richest microvascular blood supply. In mouse bone marrow, for example, a capillary network has been demonstrated at the osteomyeloid junction (Fleidner, Sandkiihler & Stodtmeister, 1956; BrBnemark, 1959; De Bruyn, Breen & Thomas, 1970). Thus, the possibility must be considered that regional differences in radioautographic labelling may reflect preferential 3H-TdR availability rather than true regional differences in cell proliferative behavior. Correspondence:Dr Stanley E. Shackney, National Cancer Institute, Building 37, Room 5A07, Bethesda, Maryland 20014, U.S.A. 363
364
S.E. Shackney, P.A . Bum, Jr and S. S.Ford
The stathmokinetic method provides an alternative to 3H-TdR studies in the study of cell population kinetic behavior. In the present study, Colcemid (Ciba) was used to explore regional kinetic differences in histologic sections of mouse bone marrow by the stathmokinetic method. The data support the conclusion that there are true kinetic differences between the subendosteal and central regions of the marrow. In addition, the data suggest differences between Colcemid's effects on myelopoiesis and its effects of thrombopoiesis. MATERIALS A N D METHODS NIH general purpose mice weighing 25-30 g were injected with Colcemid at a concentration of 2 mg/kg body weight. One group received a single injection at time 0 while the second group received injections at time 0 and every 2 hr until hour 12 for a total of seven injections. Mice were killed at intervals by perfiision fixation with neutral buffered formalin gum acacia as previously described (Shackney et al., 1975). By this method, fixative is perfused through the arterial vascular system at the time of animal sacrifice, and rapid fixation of the marrow is achieved. Four or five mice were killed at each time point. Both femurs from each animal were stripped of muscle tissue and decalcified in 10% EDTA for 7 days. The bones were then embedded in paraffin and sectioned transversely in midfemur at a thickness of 5 pm. Slides were stained by a modification of the Feulgen method. For spatial localization, a sampling band traversing the marrow at its widest point was established with the aid of an eyepiece reticule (Fig. 1). This band was subdivided into
3
7-
80pn
1_
1
Zone JI
Zone I
FIO. 1. Schematic representation of a transverse section of mouse bone marrow, showing 80 prn wide sampling band traversing the marrow and divided into sectors. The sampling band is
divided into peripheral, intermediate, and central mnes. For details, see text.
Efects of Colcemid on mouse bone marrow
365
compartments of dimensions 8 x 80 pm. In order to permit comparisons from section to section, and the pooling of data from multiple sections, the number of compartments in the sampling band was normalized to fifty, and cells were assigned to normalized compartmehts which corresponded with their relative position in the marrow sampling band in each section. To facilitate the study of differences in kinetic behavior in different regions of the marrow, the normalized sampling band was divided into three zones, as shown in Fig. 1. Zone I (compartments 1-3 and 48-50) represents the peripheral subendosteal region, Zone I1 (compartments 4-10 and 41-47) an intermediate region, and Zone I11 (compartment 1140) represents the central region of the marrow. Ten histologic sections from at least four different mice were counted at each time point. Cells were classified as immature cells, megakaryocytes, and mature granulocytes (band forms and segmental polymorphonuclear leukocytes). No attempt was made to identify cells of the erythropoietic series. RESULTS General effects The effects of a single dose and multiple doses of Colcemid on the accumulation of mitoses throughout the marrow sampling band are shown in Fig. 2(a). It is apparent that the multiple
-
c
-
e
c
e
c
c
s
0
12
24
48 Time ( hr )
FIG.2. (a) The effects of single dose (broken line) and multiple doses (solid line) of Colemidon mitoses throughout the marrow sampling band as a function of time. (b) The effects of single dose (broken line) and multiple doses (solid line) on immature cells throughout the marrow sampling band as a functionof time. (c) The effects of multiple doses of Colcemid on megalcaryocytes (solid line) and mature granulocytes (broken line) throughout the marrow as a function of time. For discussion, see text.
dose schedule produced a greater stathmokinetic effect than the single dose. The difference between the two scheduleswas apparent at 4 hr, and is attributable entirely to the second dose given at 2 hr. However, it is also apparent from Fig. 2(a) that subsequent doses of Colcemid were toxic. The rate of accumulation of mitoses decreased between 4 and 8 hr on the multiple dose schedule, and the number of cells arrested in metaphase actually fell between 8 and 12hr
S. E. Shackney,P. A . Bunn, Jr and S. S. Ford
366
during continued Colcemid administration. Degenerating metaphases were e s t observed in the marrow at 8 hr. The effects of single and multiple doses of Colcemid on immature marrow cells are shown in Fig. 2(b). There was a slight fall in immature cells following a single dose. On the multiple dose schedule, the fall in immature cells was much more pronounced. The effects of the multiple dose schedule on megakaryocytes and marrow segmented granulocytes are shown in Fig. 2(c). There was a precipitous fall in marrow granulocytes at 12 hr. In contrast, marrow megakaryocytesincreased following Colcemid administration. Regional stathmokinetic effects The effects of the multiple dose schedule of Colcemid on the immature cells in each zone are shown in Fig. 3(a). Cell loss was apparent at 4 hr in all three zones; it was greater in Zone T
120
-
( 0 )
----_-zone II --- Z O W I I I
20
0
12
24
48
12
24
48
Time ( N )
FIG.3. (a) The effectsof multiple doses of Colcemid on immature cells as a functionof time, by marrow zone. (b) The effects of multiple doses of Colcemid on mitoses as a function of time, by marrow zone. For discussion, see text.
I1 than in Zone I (P< 0.025, Student t test), and greater in Zone I11 than in Zone I1 (P< 0*0005).
The regional accumulation of mitoses can be examined in several ways. Since immature cell loss varied both spatially and temporally it is clear that the mitotic index (mitoses/ immature cells) cannot be used as a valid parameter for comparing regional mitotic differences as a function of time. The regional accumulations of mitoses per unit area, expressed relative to pretreatment values, are shown for each zone in Fig. 3(b). At 4 hr there was an eleven-fold increase in Zone I, an eight-fold increase in Zone 11, and a six-fold increase in Zone 111. The differences between Zones I and 11, and Zones I1 and 111, are both statistically significant (P< 0.01 and P < 0005, respectively).
Efects of Cokemid on mouse bone marrow
367
DISCUSSION The requirements of an ideal stathmokinetic agent have been considered in detail by Dustin (1959): (1) it should arrest cell mitoses rapidly after injection; (2) mitoses must accumulate at a constant rate over some finite time; and (3) the drug must not destroy any cells. Unfortunately, all currently available stathmokinetic cells exhibit non-ideal properties to a greater or lesser degree, necessitating the judicious choice of experimental conditions, and circumspection in drawing quantitative conclusions from the data. In choosing optimal conditions for stathmokinetic analysis we might first consider the effects of a single dose of colchicine or Cokemid. In the rat and mouse complete mitotic collection can be achieved during the first 2-3 hr after drug administration, as judged by the absence of anaphases during this interval (Dustin, 1959; Stevens Hooper, 1961; Clarke, 1971). The first reappearance of anaphases and telophases has been observed as early as 2-5-3 hr after a single dose in some studies (Smith, Thomas & Riches, 1974) and as late as 6 hr or longer in other studies (Leblond & Stevens, 1948; Dustin, 1959). Complete mitotic collection can be achieved during the first 2-3 hr over a dose range of 1-4 mg for both colchicine and Colcemid (Stevens Hooper, 1961; Clarke, 1971; Smith et al., 1974). Tannock’s contrary view that mitotic collection is incomplete over this dose range is based on dose response data obtained 4 hr after drug administration (Tannock, 1967). The appearance of degenerating metaphases in significant numbers has been observed as early as 3.5 hr after a single dose (Clarke, 1971), but in other published studies significant numbers of degenerating metaphases have been seen at times later than 4 hr (Leblond & Stevens, 1948; Morris, 1967). In the present study degenerating metaphases were seen at 8 hr but not at 4 hr. It would appear, then, that the optimal interval between the administration of a single dose of Colcemid and the collection of stathmokinetic data is in the range of 2-3 hr. This interval represents only a small fraction of the expected cell cycle time, and it is to be expected that only a small fraction of the proliferating cells will accumulate in mitosis over so short a period. In order to detect what might be relatively small regional differences in marrow proliferative rate using the stathmokinetic method, it would be advantageous to extend the period of complete metaphase collection for Colcemid for as long as possible beyond 2 hr. In the present study, injections of 2 mg/kg every 2 hr proved far more effective at 4 hr than a single 2 mg/kg injection. It would appear that each of the first two injections produced maximal stathmokinetic effects and that the effects of both doses were cumulative. Toxicity was also cumulative, but degenerating metaphases did not appear until after 4 hr. Thus, the 4 hr time point on the multiple dose schedule was chosen as the one likely to provide the most discriminating yet reliable stathmokinetic information. It is clear that the accumulation of mitoses during the first 4 hr of Colcemid administration is greater in the peripheral region of the marrow than in the central region. Thus, the present study supports previous radioautographic studies in mocse bone marrow which suggest that the rate of cell proliferation is higher in the subendosteal region than in the central region (Shackney et al., 1975). Studies on the microvasculature of the marrow have shown that there is a true capillary network at the osteomyeloid junction (Fleidner et al., 1956; BrAnemark, 1959; De Bruyn et nl., 1970). It is possible, then, that the greater accumulation of mitoses in the subendosteal
368
S. E. Shuckney, P.A . Bunn, Jr and S. S. Ford
region may have been due to preferential availability of Colcemid in this region. However, the presence of significant interphase cell loss in the central region at 4 hr indicates t h a t effective drug concentrations were achieved early throughout the marrow. The demonstration of early interphase cell loss in the present study points to difficulties inherent in the stathmokinetic method that have not received wide attention in the past. Such previously described undesirable effects as incomplete mitotic collection, mitotic degeneration, and the retardation of interphase cell progression through the cycle and reduction in the rate of DNA synthesis (Hell & Cox, 1963; Fitzgerald & Brehaut, 1970), all tend to lower the numerator of the mitotic index spuriously, leading to underestimates of population proliferativerate. Interphasecell loss, on the other hand, lowers the denominator of the mitotic index, leading to overestimates of population proliferative rate. It is quite possible that counterbalancing effects might produce a pseudo-linear increase in mitotic index with time which might bear little relation to population proliferative rate. In order to avoid the potentially confusing effects of interphase cell loss, the mitotic index was avoided as a stathmokinetic parameter in the present study, and mitotic accumulation was evaluated as mitoses per unit area in relation to pretreatment values in each region. One can draw inferencesregarding relative rates of cell proliferation by region from such data, but quantitative estimates of population proliferative rate cannot be obtained. It is of some interest that cell loss was actually greater in the central region than in the subendosteal region (Fig. 3a). Mature granulocytes are normally more abundant in the central region of the marrow than in tbe periphery (Weinbeck, 1938; Shackney et al., 1975), and serial radioautographicstudies have suggested that there is normally a centripetalmigration of myelopoieticcells which accompaniesendstage granulocyte differentiation(Shackney et al., 1975). The increased loss of immature cells from the central region and the temporal delay in the loss of mature granulocytes in comparison with the immature cells (compare Figs. 2c and 3a) suggest an interruption in the centripetal flow of proliferating cells progressing through the myeloid maturational sequence, with effects on endstage granulocyte production that become apparent when the preformed pool of mature granulocytes is depleted. The behavior of marrow megakaryocytesduring and after Colcemid administrationstands in contrast with that of the mature granulocytes. Marrow megakaryocytes increased at a time when the number of mature granulocytes was falling. Recent studies in this laboratory have confirmed a dose and schedule dependent Colcemid induced stimulatoryeffect on megakaryocyte production and have demonstrated corresponding increases in the peripheral platelet count (P.A. Bunn, S. E. Shackney and S. S. Ford, in preparation). In these respects Colcemid is similar to vincristine and vinblastine (Rak, 1972; Klener, Donner & Houskova, 1972), but Colcemid appears to be less toxic than the vinca alkaloids (unpublished observations). In any case, the preferential effects of mitotic spindle inhibitors on megakaryocyte production would suggest that these drugs may be useful not only for their stathmokinetic properties, but as pharmacologic tools in the study of differentiation in hematopoietic tissues. REFERENCES &RUM,
K. (1968) Pattern of cell production and cell migration in mouse thymus studied by autoradiography.
S c a d . J. Haemat. 5,339. B~RUM, K. (1973) Cell kinetics in mouse thymus studied by simultaneoususe of 3H-thymidineand colchicine. Cell Tissue Kinet. 6,545.
Egects of’Colcemid on mouse bone marrow
369
BR~NEMARK, P.4. (1959) Vital microscopy of bone marrow in rabbit. Scand. J. clin. Lab. Invest. 11, 38. CLARKE, R.M. (1971) A comparison of metaphase arresting agents and tritiated thymidine autoradiography in measurement of the rate of entry of cells into mitoses in the crypts of Lieberberkiihn of the rat. Cell Tissue Kinet. 4,263. DE BRUYN,P.P.H., BREEN, P.C. &THOMAS, T.B. (1970) The microcirculation of the bone marrow. Anat. Rec. 168,55. DIDERHOLM, B. & HELLMAN, B. (1960) The cell renewal in the rat adrenals studied with tritiated thymidine. Actapath. rnicrobiol. s c a d . 49,82. DUSTIN,P., JR (1959) The quantitative estimation of mitotic growth in the bone marrow of the rat by the stathmokinetic (Colchicine) method. Kinetics of Cellular Proliferation (ed. by F. Stohlman, Jr), p. 50. Grune & Stratton, New York. FITZGERALD, P.H. & BREHAUT, L.A. (1970) Depression of DNA synthesis and mitotic index by colchicine in cultured human lymphocytes. Exp. Cell Res. 59,27. FLEIDNER, T., SANDKUHLER, S. & STODTMEISTER, R. (1956) Untersuchung uber die Gefassbarchitektonik des Knochenmarkes der Ratter. Z. Zellforsch. mikrosk. Anat. 45,328. FORD, J.K. & YOUNG,R.W. (1963) Cell proliferation and displacement in the adrenal cortex of young rats injected with tritiated thymidine. Anat. Rec. 146,125. GRISHAM, J.W. (1962) A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-H3. Cancer Res. 22,842. HANNA,M.G., JR (1964) An autoradiographic study of the germinal center in spleen white pulp during early intervals of the immune response. Lab. Invest. 13,95. HELL,E. & Cox, D.G. (1963) Effects of colchicine and Colcemid on synthesis of deoxyribonucleic acid in the skin of the guinea pig’s ear in uitro. Nature, 197,287. Ism, Y., MORI.M. & ONOE,T. (1972) Studies on the germinal center. IV. Autoradiographic study of lymph node germinal centers in relation to zonal differentiation. J. Reticuloendothel. SOC.11, 383. IVERSEN, O.H., BERKNES,R. & DEW, F.(1968) Kinetics of cell renewal, cell migration and cell loss in the hairless mouse dorsal epidermis. Cell Tissue Kinet. 1,351. KLENER,P., DONNER,L. & HOUSKOVA, J. (1972) Thrombocytosis in rats induced by vinblastine. Hemostasis, 1,73. LEBLOND,C.P., GREULICH, R.C. & PEREIRA, J.P.M. (1964) Relationship of cell formation and cell migration in the renewal of stratified squamous epithelia. Ado. Biol. Skin, 5 , 39. LEBLOND, C.P. & STEVENS, C.E. (1948) The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100, 357. MORRIS,W.T. (1967) In uiuo studies on the optimum time for the action of colchicine on mouse lymphoid tissues. Exp. Cell Res. 48,209. QUASTLER, H. & SHERMAN, F.G. (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res. 17,420. RAK,K. (1972) Effect of vincristine on platelet production in mice. Brit. J. Haernat. 22, 617. SHACKNEY, S.E., FORD, S.S. & W ~ GA.B. , (1975) Kinetic-microarchitectural correlations in the bone marrow of the mouse. Cell Tissue Kinet. 8,503. SMITH,S.R., THOMAS, D.B. & Rrcm, A.C. (1974) Cell production in tumor isografts measured using Vincristine and Colcemid. Cell Tissue Kinet. 7,529. HOOPER, C.E. (1961) Use of colchicinefor the measurement of mitotic rate in the intestinal epithelium. STEVENS Amer. J. Anat. 108,231. TANNOCK, I.F. (1967) A comparison of the relative efficiencies of various interphase arrest agents. Exp. Cell Res. 47,345. TANNOCK, I.F. (1968) The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Brit. J. Cancer, 22,258. WEINBECK, J. (1938) Die Granulopoese des kindlichen Knochenmarkes und ihre Reaktion auf Infectionen. Beitr. Path. Anat. 101,268. WRIGHT, N.A. (1971) Cell proliferation in the prepubertal male rat adrenal cortex: an autoradiographic study. J. Endocrin. 49, 599.
34
T H E EFFECTS O F COLCEMID O N M O U S E B O N E MARROW STANLEY E. SHACKNEY, PAUL A. BUNN, JR A N D SHARONs. FORD Ofice of the Director, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland (Received 15 October 1975; revision received 15 January 1976) ABSTRACT
Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes. INTRODUCTION Preferential regional uptake of tritiated thymidine (3H-TdR) has been demonstrated radioautographically in a variety of tissues, including intestinal epithelium (Quastler & Sherman, 1959), epidermis (Leblond, Greulich & Pereira, 1964; Iversen, Bjerknes & Devik, 1968), regenerating liver (Grisham, 1962), thymus (Borum, 1968, 1973), lymphoid germinal centers (Hanna, 1964; Ishii, Mori & Onoe, 1972), adrenal (Diderholm & Hellman, 1960; Ford & Young, 1963; Wright, 1971) and certain tumors (Tannock, 1968). In recent studies in mouse bone marrow it has been shown that the subendosteal region of the marrow exhibits the highest labelling index and the largest number of heavily labelled cells 2 hr after pulse 3H-TdR exposure (Shackney, Ford & Wittig, 1975). Frequently, the areas of greatest 3H-TdR uptake are also those which are endowed with the richest microvascular blood supply. In mouse bone marrow, for example, a capillary network has been demonstrated at the osteomyeloid junction (Fleidner, Sandkiihler & Stodtmeister, 1956; BrBnemark, 1959; De Bruyn, Breen & Thomas, 1970). Thus, the possibility must be considered that regional differences in radioautographic labelling may reflect preferential 3H-TdR availability rather than true regional differences in cell proliferative behavior. Correspondence:Dr Stanley E. Shackney, National Cancer Institute, Building 37, Room 5A07, Bethesda, Maryland 20014, U.S.A. 363
364
S.E. Shackney, P.A . Bum, Jr and S. S.Ford
The stathmokinetic method provides an alternative to 3H-TdR studies in the study of cell population kinetic behavior. In the present study, Colcemid (Ciba) was used to explore regional kinetic differences in histologic sections of mouse bone marrow by the stathmokinetic method. The data support the conclusion that there are true kinetic differences between the subendosteal and central regions of the marrow. In addition, the data suggest differences between Colcemid's effects on myelopoiesis and its effects of thrombopoiesis. MATERIALS A N D METHODS NIH general purpose mice weighing 25-30 g were injected with Colcemid at a concentration of 2 mg/kg body weight. One group received a single injection at time 0 while the second group received injections at time 0 and every 2 hr until hour 12 for a total of seven injections. Mice were killed at intervals by perfiision fixation with neutral buffered formalin gum acacia as previously described (Shackney et al., 1975). By this method, fixative is perfused through the arterial vascular system at the time of animal sacrifice, and rapid fixation of the marrow is achieved. Four or five mice were killed at each time point. Both femurs from each animal were stripped of muscle tissue and decalcified in 10% EDTA for 7 days. The bones were then embedded in paraffin and sectioned transversely in midfemur at a thickness of 5 pm. Slides were stained by a modification of the Feulgen method. For spatial localization, a sampling band traversing the marrow at its widest point was established with the aid of an eyepiece reticule (Fig. 1). This band was subdivided into
3
7-
80pn
1_
1
Zone JI
Zone I
FIO. 1. Schematic representation of a transverse section of mouse bone marrow, showing 80 prn wide sampling band traversing the marrow and divided into sectors. The sampling band is
divided into peripheral, intermediate, and central mnes. For details, see text.
Efects of Colcemid on mouse bone marrow
365
compartments of dimensions 8 x 80 pm. In order to permit comparisons from section to section, and the pooling of data from multiple sections, the number of compartments in the sampling band was normalized to fifty, and cells were assigned to normalized compartmehts which corresponded with their relative position in the marrow sampling band in each section. To facilitate the study of differences in kinetic behavior in different regions of the marrow, the normalized sampling band was divided into three zones, as shown in Fig. 1. Zone I (compartments 1-3 and 48-50) represents the peripheral subendosteal region, Zone I1 (compartments 4-10 and 41-47) an intermediate region, and Zone I11 (compartment 1140) represents the central region of the marrow. Ten histologic sections from at least four different mice were counted at each time point. Cells were classified as immature cells, megakaryocytes, and mature granulocytes (band forms and segmental polymorphonuclear leukocytes). No attempt was made to identify cells of the erythropoietic series. RESULTS General effects The effects of a single dose and multiple doses of Colcemid on the accumulation of mitoses throughout the marrow sampling band are shown in Fig. 2(a). It is apparent that the multiple
-
c
-
e
c
e
c
c
s
0
12
24
48 Time ( hr )
FIG.2. (a) The effects of single dose (broken line) and multiple doses (solid line) of Colemidon mitoses throughout the marrow sampling band as a function of time. (b) The effects of single dose (broken line) and multiple doses (solid line) on immature cells throughout the marrow sampling band as a functionof time. (c) The effects of multiple doses of Colcemid on megalcaryocytes (solid line) and mature granulocytes (broken line) throughout the marrow as a function of time. For discussion, see text.
dose schedule produced a greater stathmokinetic effect than the single dose. The difference between the two scheduleswas apparent at 4 hr, and is attributable entirely to the second dose given at 2 hr. However, it is also apparent from Fig. 2(a) that subsequent doses of Colcemid were toxic. The rate of accumulation of mitoses decreased between 4 and 8 hr on the multiple dose schedule, and the number of cells arrested in metaphase actually fell between 8 and 12hr
S. E. Shackney,P. A . Bunn, Jr and S. S. Ford
366
during continued Colcemid administration. Degenerating metaphases were e s t observed in the marrow at 8 hr. The effects of single and multiple doses of Colcemid on immature marrow cells are shown in Fig. 2(b). There was a slight fall in immature cells following a single dose. On the multiple dose schedule, the fall in immature cells was much more pronounced. The effects of the multiple dose schedule on megakaryocytes and marrow segmented granulocytes are shown in Fig. 2(c). There was a precipitous fall in marrow granulocytes at 12 hr. In contrast, marrow megakaryocytesincreased following Colcemid administration. Regional stathmokinetic effects The effects of the multiple dose schedule of Colcemid on the immature cells in each zone are shown in Fig. 3(a). Cell loss was apparent at 4 hr in all three zones; it was greater in Zone T
120
-
( 0 )
----_-zone II --- Z O W I I I
20
0
12
24
48
12
24
48
Time ( N )
FIG.3. (a) The effectsof multiple doses of Colcemid on immature cells as a functionof time, by marrow zone. (b) The effects of multiple doses of Colcemid on mitoses as a function of time, by marrow zone. For discussion, see text.
I1 than in Zone I (P< 0.025, Student t test), and greater in Zone I11 than in Zone I1 (P< 0*0005).
The regional accumulation of mitoses can be examined in several ways. Since immature cell loss varied both spatially and temporally it is clear that the mitotic index (mitoses/ immature cells) cannot be used as a valid parameter for comparing regional mitotic differences as a function of time. The regional accumulations of mitoses per unit area, expressed relative to pretreatment values, are shown for each zone in Fig. 3(b). At 4 hr there was an eleven-fold increase in Zone I, an eight-fold increase in Zone 11, and a six-fold increase in Zone 111. The differences between Zones I and 11, and Zones I1 and 111, are both statistically significant (P< 0.01 and P < 0005, respectively).
Efects of Cokemid on mouse bone marrow
367
DISCUSSION The requirements of an ideal stathmokinetic agent have been considered in detail by Dustin (1959): (1) it should arrest cell mitoses rapidly after injection; (2) mitoses must accumulate at a constant rate over some finite time; and (3) the drug must not destroy any cells. Unfortunately, all currently available stathmokinetic cells exhibit non-ideal properties to a greater or lesser degree, necessitating the judicious choice of experimental conditions, and circumspection in drawing quantitative conclusions from the data. In choosing optimal conditions for stathmokinetic analysis we might first consider the effects of a single dose of colchicine or Cokemid. In the rat and mouse complete mitotic collection can be achieved during the first 2-3 hr after drug administration, as judged by the absence of anaphases during this interval (Dustin, 1959; Stevens Hooper, 1961; Clarke, 1971). The first reappearance of anaphases and telophases has been observed as early as 2-5-3 hr after a single dose in some studies (Smith, Thomas & Riches, 1974) and as late as 6 hr or longer in other studies (Leblond & Stevens, 1948; Dustin, 1959). Complete mitotic collection can be achieved during the first 2-3 hr over a dose range of 1-4 mg for both colchicine and Colcemid (Stevens Hooper, 1961; Clarke, 1971; Smith et al., 1974). Tannock’s contrary view that mitotic collection is incomplete over this dose range is based on dose response data obtained 4 hr after drug administration (Tannock, 1967). The appearance of degenerating metaphases in significant numbers has been observed as early as 3.5 hr after a single dose (Clarke, 1971), but in other published studies significant numbers of degenerating metaphases have been seen at times later than 4 hr (Leblond & Stevens, 1948; Morris, 1967). In the present study degenerating metaphases were seen at 8 hr but not at 4 hr. It would appear, then, that the optimal interval between the administration of a single dose of Colcemid and the collection of stathmokinetic data is in the range of 2-3 hr. This interval represents only a small fraction of the expected cell cycle time, and it is to be expected that only a small fraction of the proliferating cells will accumulate in mitosis over so short a period. In order to detect what might be relatively small regional differences in marrow proliferative rate using the stathmokinetic method, it would be advantageous to extend the period of complete metaphase collection for Colcemid for as long as possible beyond 2 hr. In the present study, injections of 2 mg/kg every 2 hr proved far more effective at 4 hr than a single 2 mg/kg injection. It would appear that each of the first two injections produced maximal stathmokinetic effects and that the effects of both doses were cumulative. Toxicity was also cumulative, but degenerating metaphases did not appear until after 4 hr. Thus, the 4 hr time point on the multiple dose schedule was chosen as the one likely to provide the most discriminating yet reliable stathmokinetic information. It is clear that the accumulation of mitoses during the first 4 hr of Colcemid administration is greater in the peripheral region of the marrow than in the central region. Thus, the present study supports previous radioautographic studies in mocse bone marrow which suggest that the rate of cell proliferation is higher in the subendosteal region than in the central region (Shackney et al., 1975). Studies on the microvasculature of the marrow have shown that there is a true capillary network at the osteomyeloid junction (Fleidner et al., 1956; BrAnemark, 1959; De Bruyn et nl., 1970). It is possible, then, that the greater accumulation of mitoses in the subendosteal
368
S. E. Shuckney, P.A . Bunn, Jr and S. S. Ford
region may have been due to preferential availability of Colcemid in this region. However, the presence of significant interphase cell loss in the central region at 4 hr indicates t h a t effective drug concentrations were achieved early throughout the marrow. The demonstration of early interphase cell loss in the present study points to difficulties inherent in the stathmokinetic method that have not received wide attention in the past. Such previously described undesirable effects as incomplete mitotic collection, mitotic degeneration, and the retardation of interphase cell progression through the cycle and reduction in the rate of DNA synthesis (Hell & Cox, 1963; Fitzgerald & Brehaut, 1970), all tend to lower the numerator of the mitotic index spuriously, leading to underestimates of population proliferativerate. Interphasecell loss, on the other hand, lowers the denominator of the mitotic index, leading to overestimates of population proliferative rate. It is quite possible that counterbalancing effects might produce a pseudo-linear increase in mitotic index with time which might bear little relation to population proliferative rate. In order to avoid the potentially confusing effects of interphase cell loss, the mitotic index was avoided as a stathmokinetic parameter in the present study, and mitotic accumulation was evaluated as mitoses per unit area in relation to pretreatment values in each region. One can draw inferencesregarding relative rates of cell proliferation by region from such data, but quantitative estimates of population proliferative rate cannot be obtained. It is of some interest that cell loss was actually greater in the central region than in the subendosteal region (Fig. 3a). Mature granulocytes are normally more abundant in the central region of the marrow than in tbe periphery (Weinbeck, 1938; Shackney et al., 1975), and serial radioautographicstudies have suggested that there is normally a centripetalmigration of myelopoieticcells which accompaniesendstage granulocyte differentiation(Shackney et al., 1975). The increased loss of immature cells from the central region and the temporal delay in the loss of mature granulocytes in comparison with the immature cells (compare Figs. 2c and 3a) suggest an interruption in the centripetal flow of proliferating cells progressing through the myeloid maturational sequence, with effects on endstage granulocyte production that become apparent when the preformed pool of mature granulocytes is depleted. The behavior of marrow megakaryocytesduring and after Colcemid administrationstands in contrast with that of the mature granulocytes. Marrow megakaryocytes increased at a time when the number of mature granulocytes was falling. Recent studies in this laboratory have confirmed a dose and schedule dependent Colcemid induced stimulatoryeffect on megakaryocyte production and have demonstrated corresponding increases in the peripheral platelet count (P.A. Bunn, S. E. Shackney and S. S. Ford, in preparation). In these respects Colcemid is similar to vincristine and vinblastine (Rak, 1972; Klener, Donner & Houskova, 1972), but Colcemid appears to be less toxic than the vinca alkaloids (unpublished observations). In any case, the preferential effects of mitotic spindle inhibitors on megakaryocyte production would suggest that these drugs may be useful not only for their stathmokinetic properties, but as pharmacologic tools in the study of differentiation in hematopoietic tissues. REFERENCES &RUM,
K. (1968) Pattern of cell production and cell migration in mouse thymus studied by autoradiography.
S c a d . J. Haemat. 5,339. B~RUM, K. (1973) Cell kinetics in mouse thymus studied by simultaneoususe of 3H-thymidineand colchicine. Cell Tissue Kinet. 6,545.
Egects of’Colcemid on mouse bone marrow
369
BR~NEMARK, P.4. (1959) Vital microscopy of bone marrow in rabbit. Scand. J. clin. Lab. Invest. 11, 38. CLARKE, R.M. (1971) A comparison of metaphase arresting agents and tritiated thymidine autoradiography in measurement of the rate of entry of cells into mitoses in the crypts of Lieberberkiihn of the rat. Cell Tissue Kinet. 4,263. DE BRUYN,P.P.H., BREEN, P.C. &THOMAS, T.B. (1970) The microcirculation of the bone marrow. Anat. Rec. 168,55. DIDERHOLM, B. & HELLMAN, B. (1960) The cell renewal in the rat adrenals studied with tritiated thymidine. Actapath. rnicrobiol. s c a d . 49,82. DUSTIN,P., JR (1959) The quantitative estimation of mitotic growth in the bone marrow of the rat by the stathmokinetic (Colchicine) method. Kinetics of Cellular Proliferation (ed. by F. Stohlman, Jr), p. 50. Grune & Stratton, New York. FITZGERALD, P.H. & BREHAUT, L.A. (1970) Depression of DNA synthesis and mitotic index by colchicine in cultured human lymphocytes. Exp. Cell Res. 59,27. FLEIDNER, T., SANDKUHLER, S. & STODTMEISTER, R. (1956) Untersuchung uber die Gefassbarchitektonik des Knochenmarkes der Ratter. Z. Zellforsch. mikrosk. Anat. 45,328. FORD, J.K. & YOUNG,R.W. (1963) Cell proliferation and displacement in the adrenal cortex of young rats injected with tritiated thymidine. Anat. Rec. 146,125. GRISHAM, J.W. (1962) A morphologic study of deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine-H3. Cancer Res. 22,842. HANNA,M.G., JR (1964) An autoradiographic study of the germinal center in spleen white pulp during early intervals of the immune response. Lab. Invest. 13,95. HELL,E. & Cox, D.G. (1963) Effects of colchicine and Colcemid on synthesis of deoxyribonucleic acid in the skin of the guinea pig’s ear in uitro. Nature, 197,287. Ism, Y., MORI.M. & ONOE,T. (1972) Studies on the germinal center. IV. Autoradiographic study of lymph node germinal centers in relation to zonal differentiation. J. Reticuloendothel. SOC.11, 383. IVERSEN, O.H., BERKNES,R. & DEW, F.(1968) Kinetics of cell renewal, cell migration and cell loss in the hairless mouse dorsal epidermis. Cell Tissue Kinet. 1,351. KLENER,P., DONNER,L. & HOUSKOVA, J. (1972) Thrombocytosis in rats induced by vinblastine. Hemostasis, 1,73. LEBLOND,C.P., GREULICH, R.C. & PEREIRA, J.P.M. (1964) Relationship of cell formation and cell migration in the renewal of stratified squamous epithelia. Ado. Biol. Skin, 5 , 39. LEBLOND, C.P. & STEVENS, C.E. (1948) The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100, 357. MORRIS,W.T. (1967) In uiuo studies on the optimum time for the action of colchicine on mouse lymphoid tissues. Exp. Cell Res. 48,209. QUASTLER, H. & SHERMAN, F.G. (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res. 17,420. RAK,K. (1972) Effect of vincristine on platelet production in mice. Brit. J. Haernat. 22, 617. SHACKNEY, S.E., FORD, S.S. & W ~ GA.B. , (1975) Kinetic-microarchitectural correlations in the bone marrow of the mouse. Cell Tissue Kinet. 8,503. SMITH,S.R., THOMAS, D.B. & Rrcm, A.C. (1974) Cell production in tumor isografts measured using Vincristine and Colcemid. Cell Tissue Kinet. 7,529. HOOPER, C.E. (1961) Use of colchicinefor the measurement of mitotic rate in the intestinal epithelium. STEVENS Amer. J. Anat. 108,231. TANNOCK, I.F. (1967) A comparison of the relative efficiencies of various interphase arrest agents. Exp. Cell Res. 47,345. TANNOCK, I.F. (1968) The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Brit. J. Cancer, 22,258. WEINBECK, J. (1938) Die Granulopoese des kindlichen Knochenmarkes und ihre Reaktion auf Infectionen. Beitr. Path. Anat. 101,268. WRIGHT, N.A. (1971) Cell proliferation in the prepubertal male rat adrenal cortex: an autoradiographic study. J. Endocrin. 49, 599.
34
