THE EFFECTS OF COLCEMID ON MOUSE BONE MARROW

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.     

KINETIC-MICROARCHITECTURAL CORRELATIONS IN THE BONE MARROW OF THE MOUSE

Transverse histologic sections of bone marrow obtained from mice that were sacrificed by perfusion fixation at intervals following tritiated thymidine injection were studied by means of radioautography. A kinetic gradient was demonstrated across the marrow section, with the highest proliferative rate in the subendosteal region. Megakaryocytes were shown to originate from the rapidly proliferating subendosteal cells. The immediate proliferating precursors of mature granulocytes were slowly proliferating cells found predominantly in the central region of the marrow. It was concluded that in the steady state there must be a migration of cells from the subendosteal region to the central region with concomitant growth retardation of the migrating cells.     

Kinetic-microarchitectural correlations in the bone marrow of the mouse.

Transverse histologic sections of bone marrow obtained from mice that were sacrificed by perfusion fixation at intervals following tritiated thymidine injection were studied by means of radioautography. A kinetic gradient was demonstrated across the marrow section, with the highest proliferative rate in the subendosteal region. Megakaryocytes were shown to originate from the rapidly proliferating subendosteal cells. The immediate proliferating precursors of mature granulocytes were slowly proliferating cells found predominantly in the central region of the marrow. It was concluded that in the steady state there must be a migration of cells from the subendosteal region to the central region with concomitant growth retardation of the migrating cells.     

Effect of plutonium-239 on the mitotic activity of mouse bone marrow cells

Summary Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq²³⁹Pu kg^–1. The attention was turned to the femoral midshaft and the mitose concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.     

Ultrastructure of hematopoietic and stromal cells of the subendosteal region of the bone marrow

Using electron microscopic cytochemistry and immunoelectron microscopy, the ultrastructure of bone marrow (BM) cells of the subendosteal region with a high colony-forming (CFUs) ability was studied. In comparison with the central part of BM, the subendosteal region of CBA and BALB/c mice contains a higher number of lymphocyte-like mononuclears, bearing an antigen, common with the brain surface one but negative for peroxidase and acid and alkaline phosphatase. The ultrastructure of these cells is similar to that of presumptive hematopoietic stem cells. In the subendosteal region mononuclears are concentrated with the lower nucleo-cytoplasmic ratio, a feston-like line of the nucleus and more numerous organoids. These cells are characteristic of BM myeloid islands composed of granulocytes being on various stages of differentiation, and of reticular cells positive for alkaline phosphatase.     

Architecture of the marrow vasculature in three amphibian species and its significance in hematopoietic development.

Architecture of the bone marrow vasculature, particularly that of the femur, was analyzed in three amphibian species in relation to the early phylogeny of marrow hematopoiesis. A dye-injection method and histological techniques, including both serial sectioning and reconstruction methods, were used for this purpose. From these observations the following conclusions may be drawn. (1) Marrow hematopoiesis is absent from the femur of the urodelan (Triturus pyrrhogaster) and appears first in the femur of the primitive anuran (Xenopus laevis) (2) The site of primitive hematopoiesis (granulopoiesis) is the subendosteal region where the venous vascular net develops. (3) The primitive vascular architecture observed in the femur of Xenopus is characterized by the absence of a central vein. Subendosteal veins drain the blood from the bone marrow. A vein collateral to the primary artery appears in the femur of Rana catesbeiana, an advanced anuran, in which further development of both the subendosteal venous plexus and hematopoietic activity are noted. In both anura examined, the primitive blood sinuses form near the mid-shaft of the femur. The proliferation of mesenchymal elements containing dark pigment, presumably melanin, was also noted in this area. (4) The architecture of marrow vessels in Rana approaches the structure noted in mammalian bone marrow. (5) Fat tissue is observed in the urodelan bone marrow prior to the appearance of hematopoietic activity. This indicates that the formation of marrow fat is phylogenetically unrelated to the development of hematopoiesis. The present investigation on primitive hematopoiesis suggests that the development of hematopoietic activity is intimately related to the development of the marrow vasculature, particularly that of the subendosteal venous plexus. A favorable vascular arrangement may be necessary to allow active hematopoiesis.     

An in situ study of B-lymphocytopoiesis in rat bone marrow. Topographical arrangement of terminal deoxynucleotidyl transferase-positive cells and pre-B cells

To understand bone marrow (BM) as a site of B-lymphocytopoiesis, insight into the topographical arrangement of developing B cells and their relationships to the microenvironment in vivo is required. To study the spatial distribution of B lymphocyte progenitors defined by intracellular markers (cytoplasmic mu H chain and nuclear terminal deoxynucleotidyl transferase (TdT], we developed a technique to cut frozen femurs of rat, yielding cross-sections with intact subendosteal and central marrow. By using (double) immunofluorescence staining techniques we located pre-B and TdT+ cells, and IgM+ B cells in those sections. Of the B cells present in BM, one-third was accumulated in the lumen of blood sinuses. The rest were in the BM parenchyma, as were virtually all pre-B and TdT+ cells. The subendosteal area was twice as rich in pre-B and TdT+ cells as the central area, and within the subendosteal area a profound positive gradient toward the bone was evident. B cells showed an equal distribution over the center and the periphery of the BM. The distribution patterns of B lineage cells in the BM parenchyma were analyzed and shown in part to deviate from random distribution. Additional study of clonal development and microenvironmental factors in hematopoiesis will have to clarify the underlying mechanisms for the observed distribution patterns of B cell precursors in BM.     

Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the "quiescent" and "proliferative" niches in which hematopoietic stem cells and progenitors reside.     

In situ visualization of hemopoietic cell subsets and stromal elements in rat and mouse bone marrow by immunostaining of frozen sections

We have developed a method to section frozen long bones of rat and mouse and stained bone marrow (BM) by (double) immunofluorescence and immunoperoxidase. Here we report this method and reveal the location of early hemopoietic progenitors (Thy-1) and myeloid cells (Mac-1) in mouse BM, and early hemopoietic progenitors and lymphoid cells (Thy-1), erythroid cells (HIS49), and macrophages (ED2) in rat BM. In mouse BM our new findings include (a) the scattered localization of early hemopoietic progenitors (Thy-1low) all over the marrow, and (b) the presence of Thy-1+ stromal cells, mainly subendosteally. In rat BM an important finding is that of (a) a subendosteal region of 12-14 hemopoietic cell layers characterized by an abundance of Thy-1 and the virtual absence of erythroid cells, and (b) the scattering of Thy-1very bright cells which are candidates for the earliest hemopoietic progenitors in this species. The results illustrate that the technique is an excellent tool for studying the topology of BM as an organ of hemopoiesis.     

SHORT-TERM CULTURES OF MOUSE MARROW CELLS SEPARATED BY VELOCITY SEDIMENTATION

Our experiments were designed to identify the source of the increased number of cells forming colonies in culture (CFU-C) detected after short-term culture of mouse marrow cells over 'feeders' of renal tubules. Accordingly, marrow cell suspensions were fractionated by velocity sedimentation and aliquots of each fraction cultured for 2 days over tubule 'feeders'. We found a greater increase of CFU-C in suspensions of slowly sedimenting cells as compared to rapidly sedimenting cells. Colcemid and vinblastine were used to alter the peak sedimentation velocity of marrow suspensions by changing the distribution of cells in the cell cycle. After such manipulations, the peak increase in CFU-C continued to be associated with fractions containing slowly sedimenting cells. Such fractions also contained most of the pluripotent stem cells identified by the spleen colony technique.     

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.     

CHROMOSOME PULVERIZATION IN HUMAN BINUCLEATE CELLS FOLLOWING COLCEMID TREATMENT

Under the influence of Colcemid, a substantial number of binucleate human cells from a line infected with herpes-like virus was found to possess pulverized chromosomes. Although this abnormality was also detected in untreated binucleate cells, the increase in the number of pulverized cells after the addition of Colcemid was too striking to be explained by accumulation of spontaneously occurring cells in response to the mitotic inhibition by Colcemid. Furthermore, the induction of pulverization may be dependent upon Colcemid concentration. These findings imply an involvement of Colcemid in the mechanism of pulverization induction in the system studied. When tritiated thymidine was added to the culture medium simultaneously with Colcemid, the majority of binucleate cells with an intact and a pulverized chromosome set incorporated this isotope into the pulverized set only. This obviously suggests that the nuclei in the binucleate cell are asynchronous in DNA synthesis, and that this asynchrony is intimately related to the induction of the pulverization phenomenon. It seems very probable that the late S phase in the late synthesizing nuclei represents a critical stage at which damage to the chromosomes most readily occurs.     

A COMPARISON OF METAPHASE ARRESTING AGENTS AND TRITIATED THYMIDINE AUTORADIOGRAPHY IN MEASUREMENT OF THE RATE OF ENTRY OF CELLS INTO MITOSIS IN THE CRYPTS OF LIEBERKÜHN OF THE RAT

Three methods of estimating cell production rate were used: the rate of accumulation of metaphases blocked by Colcemid, or by vinblastine, and the rate of increase of labelled nuclei after administration of tritiated thymidine. These rates were determined for three sites in the small intestine by counts made on whole micro-dissected crypts, fixed at various times after the administration of the agents.
Within the limits of error of the methods, the cell production rate per crypt was the same when measured by each method (35/hr), and showed a slight fall from the proximal to the distal end of the small intestine (36/hr to 33/hr). the advantages and limitations of each method are discussed.     

Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.     

The fate of epidermal colcemid-arrested mitoses

This study reports the fate of hairless mouse epidermal basal cells arrested in mitosis by a traditional stathmokinetic dose of 0.15 mg Colcemid. Epidermal basal cells in the S phase were labeled with 30 microCi (3H)TdR i.p. After 1 h, four animals from a cage of eight mice were given 0.15 mg Colcemid (Fluka) in 0.5 ml saline, and the other four mice were given saline only. Groups of eight mice (four experimental, four controls) were sacrificed 4, 9, 13, 21 and 25 h after (3H)TdR injection (i.e. 3, 8, 12, 16, 20 and 24 h after Colcemid). The following cell kinetic parameters were determined: the number of labeled basal and suprabasal cells, the mean grain count of the labeled cells, the specific activity, the mitotic count, the number of labeled mitoses, the fraction of labeled mitoses curve and the fraction of cells in S and in G2 as determined by flow cytometry. "Labeled paired twins", i.e. adjoining labeled cells with approximately the same grain count, were also scored. All the results taken together support the conclusion that cells labeled with (3H)TdR and arrested 1 h later with 0.15 mg Colcemid go through at least one subsequent cell division and thereafter some of them move out into the suprabasal layer at a normal rate. Hence, after this dose of Colcemid, cells arrested in mitosis for some hours do not die, and the Colcemid treatment does not seem to produce hyperploid cells. The study confirms the usefulness of this dose of Colcemid as a convenient tool for cell kinetic studies.     

Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. I. Light microscope observations

Cytochalasin B (CB) induces a biphasic retraction is some cell types. The rapid response that peaks in 30 min leads to the "dendritic" condition. Replicating myogenic and fibrogenic cells, as well as postmitotic myoblasts and myotubes, participate in this reaction. This is followed by a slower phase that requires 40 h for stabilization and leads to the fully "absorized" state. Only replicating myogenic and fibrogenic cells participate in this reaction. Postmitotic myoblasts and myotubes do not arborize but round up and float off into the medium. Pretreatment with Colcemid does not block the rapid response to CB, but does block arborization. CB-arborized cells exposed to Colcemid while in the presence of CB develop sufficient tension to pull themselves apart. If CB depolymerizes actin-like filaments, and if such filaments constitute the only contractile system in the cell, then it is difficult to visualize how cells in CB develop such tension. Colcemid induces twisting, birefringent bands in interphase- and metaphase-arrested myogenic and fibrogenic cells, and in postmitotic myotubes. Such bands are more evident when CB-arborized cells are removed from CB and allowed to relax in Colcemid. These birefringent bands assemble in the prescence of cycloheximide, and may constitute 20% of the volume of the cell.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

A ROLE FOR MICROTUBULES IN THE REGULATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE AND CHOLESTEROL BIOSYNTHESIS IN CULTURED GLIAL CELLS 1

—Cultured C-6 glial cells were utilized to evaluate the effect of the antimicrotubular drug, Colcemid, on 3-hydroxy-3-melhylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis in cultured C-6 glial cells. The data indicate that Colcemid causes a marked inhibition of cholesterol synthesis (from [¹⁴C]acetate or ³H₂O) in these cells. A concentration of 0.5 μM led to a 50% lower rate of synthesis after 2 h and an 80–85% lower rate after 12 h or longer. That the effect of Colcemid is mediated at the level of HMG-CoA reductase was shown by defining closely coordinate temporal and quantitative changes in the activity of this enzyme under identical conditions. No comparable change in cell growth or in total protein synthesis accompanied the effect of Colcemid. The drug did lead to a decrease in the rate of DNA synthesis (from [³H]thymidine) but this effect was preceded by the decrease in the rate of cholesterol synthesis. Marked changes in glial cell shape were induced by exposure to Colcemid, and the temporal and quantitative aspects of these changes appeared to closely parallel the effects on reductase activily and cholesterol synthesis. The dala suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in mammalian cells and that there are important interrelations between microtubules, glial differentiation and cholesterol synthesis.     

Effect of mitochondrial inhibitors on metaphase-telophase progression and nuclear membrane formation in Chinese hamster cells

Chinese hamster Don cells in log-phase were exposed to Colcemid during the G2 period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. An inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca2+, stimulated the formation of micronuclei. This study indicates that Ca2+ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca2+ sequestration in the mitochondria and chelation of Ca2+ by EGTA as dominant factors.