A cellular analysis of residual hemopoietic deficiencies in mice after 4 repeated doses of 4.5 gray X rays

A sub-optimal plateau in numbers of femoral stem-cells (CFU-S) in mice after 4 doses of 4.5 Gray X rays (each separated by 21 days), was shown to persist at 20–30 % of control up to 1 year after the last dose, when about 50 % of the mice had survived. The concentration of white cells in the blood was maintained persistently at about 70% of control, whereas the concentration of red cells was normal up to 4 months and then it declined to about 75% of control at 10 months after irradiation. Concentrations of some committed progenitor cells in the marrow (GM-CFC and ERC), which are capable of amplification cell divisions, were intermediate between the concentrations of marrow stem cells and mature blood cells in both the granuloid and the erythroid cell lineages, respectively. Hence increased amplification was a mechanism operating for a prolonged period in the production of numbers of mature cells. The numbers were subnormal, however, and this corresponded to only 1 extra amplification division on average.There was a slow decline after 6 months in the numbers of CFU-S, BFU-E and GM-CFC, and in the hematocrit, with reference to age-matched controls. The decline was due partly to a prevention of the natural increase in cell numbers in the marrow with the age of the mice, which was also seen with the femoral content of a stromal progenitor cell (CFU-F). A defect in the repeatedly-irradiated CFU-S population was detected as a persistent inability to produce colonies containing the same number of daughter CFU-S as contained in colonies derived from unirradiated marrow and assayed at the same time.     

Long-term bone-marrow damage in children treated for ALL: evidence from in vitro colony assays (GM-CFC and CFUF).

We have studied granulocyte-macrophage progenitor cells (GM-CFC) in serial bone marrow aspirates from 43 children who had been treated for acute lymphoblastic leukaemia (ALL). All patients were in full remission, not receiving anti-leukaemic therapy and 42 out of the 43 had normal peripheral blood counts. Thirty-seven patients have received standard amounts of chemotherapy and 6 have received additional therapy for relapses occurring in the first treatment-free interval. In the former group estimation of GM-CFC incidence did not provide evidence of long-term residual bone-marrow damage. In the latter, however, the mean incidence of GM-CFC was significantly reduced. This reduction was also apparent when the incidence of GM-CFC was related to the incidence of non-haemopoietic progenitor cells within the marrow (CFU-F).     

4-Hydroperoxycyclophosphamide inhibits proliferation by human granulocyte-macrophage colony-forming cells (GM-CFC) but spares more primitive progenitor cells

Despite its considerable toxicity to haemopoietic colony-forming cells, 4-hydroperoxycyclophosphamide (4-HC) has successfully been used to purge marrow of leukaemic cells before it is used to rescue patients from high-dose chemoradiotherapy. These conflicting observations indicate that haemopoietic progenitor cells that are not detected by the established colony-forming assays survive exposure to 4-HC and repopulate the marrow. The recent finding that murine spleen colony-forming cells (CFU-S) are resistant to 4-HC [Porcellini A, et al. (1983) Expl Hemat. 11 (suppl 14) 331 (abstract)] [14] also indicates that sensitivity to 4-HC can be used to distinguish primitive progenitor cells from committed progenitor cells. As part of a study on the nature of a population of blast colony-forming cells in human bone marrow, we tested their sensitivity to 4-HC to see whether they also are spared by the drug. We found that 4-HC had much less effect on the blast colony-forming cells than on the granulocyte-macrophage colony-forming cells (GM-CFC). This result suggests that the blast-colony-forming cells may be early human haemopoietic progenitor cells.     

Some effects of chemotherapeutic drugs

Summary cis-Platinum is a relatively new active anticancer drug. In the study described in this paper, its toxicity was tested in the hematopoietic and renal systems of mice after six injections of 3 mg per kg body weight at 10-day intervals.Acute hematopoietic toxicity was studied by determining the survival of pluripotent (CFU-S) and granulo-macrophagic unipotent (GM-CFC) stem cells. The number of nucleated cells in the bone marrow and in the spleen and the number of granulocytes in the blood were determined.Renal toxicity was studied by histological examination of kidneys from treated mice compared with control animals.The number of stem cells in the bone marrow and in the spleen decreased during the treatment. One year after treatment, the autorepopulating ability of CFU-S was still diminished in spite of normal numbers of these cells.No renal damage could be demonstrated by light microscopy when the protocol described was used.Abbreviations used in this paper CFU-S pluripotent hemopoietic stem cells assessed by the spleen colony technique - GM-CFC granulo-macrophagic progenitor cells - BFU-E erythroid progenitor cells - E/G ratio ratio of erythroid and granulocytic colonies in the recipient spleen and assessed by histological examination - Ara-C cytosine arabinoside     

Synergistic interactions allow colony formation in vitro by murine haemopoietic stem cells

A clonogenic assay for cells that give rise to macroscopic colonies in agar or methyl cellulose cultures using untreated, normal murine bone marrow as a source of stem cells is described. We have characterized the clonogenic cell, which has been designated CFU-A, by comparing its properties with those of multipotential stem cells (assayed as CFU-S) and lineage-restricted progenitor cells (assayed as GM-CFC). The investigations have included assessment of proliferative status and response to CFU-S proliferation regulators, response to 5-fluorouracil, buoyant cell density, radial distribution in the femur and response to ionizing radiation. We conclude that the CFU-A has properties in common with CFU-S that differ from those of GM-CFC. The data are consistent with the CFU-A assay detecting part of the multipotential stem cell population also detected by spleen colony formation.     

The cellular specificity of haemopoietic stem cell proliferation regulators

The range of specificity of the CFU-S proliferation inhibitor and stimulator which are produced endogenously in the bone marrow has been investigated by measuring their effects on the proportion of cells killed by tritiated thymidine in mixed colony- (CFC-mix), erythroid burst- (BFU-E) and granulocyte/macrophage colony- (GM-CFC) forming cells as well as spleen colony forming units (CFU-S). Both CFU-S and CFC-mix were triggered by the stimulator into DNA-synthesis but BFU-E and GM-CFC were unaffected. The range of activity of the inhibitor was confined solely to the CFU-S population. This defined the specificity of both inhibitor and stimulator for the multipotent cells. The differential sensitivity of CFU-S and CFC-mix to the inhibitor and the lack of it for the stimulator suggested (a) that the CFC-mix is a relatively mature subpopulation of the CFU-S compartment and (b) that the relative sensitivity of a CFU-S to these factors changes as it matures from the early stem cell stage (Inhibitor-sensitive) to the more mature stages (Stimulator-sensitive) before becoming committed to a specific line of differentiation. The specificity of the inhibitor for haemopoietic stem cells suggests its potential value during chemotherapeutic procedures.     

Regulation of murine granulocyte-macrophage progenitor cell and haemopoietic stem cell proliferation by factors produced in human fetal liver

At early stages (11–14 weeks) of gestation in human fetal liver few granulocyte-macrophage colony-forming cells (GM-CFC) are in DNA synthesis, whereas later in gestation (> 14 weeks) a large proportion of GM-CFC are in S-phase [Moore M.A.S. & Williams N. (1973) Cell Tissue Kinet.6, 461].Incubation of normal murine bone marrow GM-CFC (approx. 40% in DNA synthesis) with a supernatant from an early human fetal liver (11–14 weeks), reduced the proportion synthesizing DNA to <5%. In contrast, the proportion of murine GM-CFC synthesizing DNA was not affected by incubation with a supernatant from a late fetal liver (> 14 weeks).GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant.It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle. In normal murine bone marrow (NMBM) approx. 10% of the haematopoietic stem cells (CFU-S) are synthesizing DNA. The proportion of CFU-S synthesizing DNA was increased to approx. 40% by incubation with a human fetal liver supernatant from all gestational ages tested (11–18 weeks).The specificity of these CFU-S and GM-CFC proliferation regulators is well demonstrated by an early gestation human fetal liver supernatant which will stimulate CFU-S proliferation but inhibit GM-CFC proliferation.The inhibitor and stimulator of GM-CFC proliferation are both produced by non-adherent human fetal liver cells. The GM-CFC proliferation inhibitor has a mol. wt of > 100,000 and the stimulator a mol. wt of 30,000–50,000. In contradistinction, the CFU-S proliferation stimulator is produced by adherent human fetal liver cells and has a mol. wt of 30,000–50,000.     

Comparison of the sensitivity of normal and leukaemic myeloid progenitors to in-vitro incubation with cytotoxic drugs: a study of pharmacological purging

The sensitivity of myeloid leukaemic colony forming cells (AML-CFC), to five cytotoxic drugs has been compared in two culture systems with the sensitivity of normal myeloid progenitor cells (GM-CFC). No increased sensitivity was found for AML-CFC to any of the chemotherapeutic agents studied. AML-CFC were significantly less sensitive than normal GM-CFC to mafosfamide at the doses commonly used to purge bone marrow autografts. It is suggested that AML cells probably display similar sensitivity to cytotoxic agents as normal myelopoietic cells at a similar stage of differentiation. Hence complete elimination of the leukemic clone by pharmacological purging may be incompatible with bone marrow re-engraftment. We conclude that purging AML autografts with any of the agents examined has little scientific basis.     

A cellular analysis of long-term haematopoietic damage in mice after repeated treatment with cyclophosphamide

Summary Following repeated treatment of mice with cyclophosphamide (5×200 mg/kg) it was found that slight, but significant, residual marrow damage persisted for at least half the lifespan of the animals. This long-term damage occurred despite preferential sparing of those multipotential haematopoietic cells (CFU-S) that had a high self-renewal capacity after each step of the multistep regimen and despite a smaller CFU-S kill after each successive dose. The damage was characterized by low mean numbers of CFU-S and stromal colony-forming cells (CFU-F) which were around 70% of control values.Examination of individual animals revealed that the majority had slightly subnormal numbers of CFU-S and CFU-F, with only a few suffering a more severe injury, including 8% of mice with clinical hypoplasia or myelodysplasia.     

Effect of adult thymectomy and splenectomy on pluripotent stem cells and progenitors of the myeloid and B-lymphoid lineages

In order to investigate the regulatory interactions which occur between the bone marrow, the thymus and the spleen during hemopoiesis, the numbers of granulo-macrophage and B-lymphocyte precursors (GM-CFC and BL-CFC) and the kinetics of pluripotent stem cells (CFU-S) have been studied in the bone marrow and the spleen of normal adult thymectomized and/or adult splenectomized mice with or without T-dependent antigen stimulation. The results suggest that (1) medullary and splenic CFU-S may be two different populations of the stem cell compartment; (2) the thymus is involved in medullary but not in splenic CFU-S proliferation in response to T-antigen challenge; (3) the spleen influences GM-CFC and BL-CFC numbers and (4) antigenic stimulation modifies medullary BL-CFC.     

Differential seeding of injected hemopoietic precursor cells in the bone marrow and spleen of irradiated mice

The seeding efficiency was determined of syngeneic granulocyte, macrophage, erythroid/mixed and megakaryocyte colony-forming cells (G-GFC, GM-CFC, M-CFC, E/Mix-CFC, MEG-CFC) in the femoral bone marrow and spleen of lethally-irradiated C57BL mice. The overall seeding efficiency of CFC's was similar to that for multipotential stem cells (CFU-S) in the marrow but in the spleen CFC seeding efficiency was ten-fold lower than for CFU-S. Two and a half hours after transplantation of 10⁷ bone marrow cells, the relative frequencies of E/Mix-CFC's and M-CFC's recovered from the recipient marrow were higher than in the injected marrow population. However the relative frequencies of CFC's recovered from the spleen corresponded closely to those of the injected marrow population.     

Macrophage inflammatory protein 1alpha attenuates the toxic effects of temozolomide in human bone marrow granulocyte-macrophage colony-forming cells.

Macrophage inflammatory protein 1alpha (MIP-1alpha) is a chemokine that may act principally by preventing hemopoietic cells from entering G1, thereby attenuating the cytotoxic effects of cell cycle-specific chemotherapeutic agents. Here we examine the effect of MIP-1alpha on the sensitivity of human granulocyte-macrophage hemopoietic progenitor cells (granulocyte-macrophage colony-forming cells; GM-CFCs) with the cytotoxic effects of antitumor agents that act mainly via alkylation at the O6 position of guanine in DNA. Mononuclear cell preparations from human bone marrow were used in an in vitro GM-CFC colony-forming assay. The GM-CFC survival from individual patients displayed a range of sensitivities to the methylating agent temozolomide [(Tz) 20-55% survival at 10 microg/ml Tz]. However, in all 16 cases, MIP-1alpha (50 ng/ml) protected against GM-CFC killing: survival in the presence of MIP-1alpha ranged from 65-97% at 10 microg/ml Tz, with GM-CFCs being 1.5-4.5-fold more resistant than control cells from the same patient. The highest levels of protection were seen in the GM-CFCs with the highest sensitivity in the absence of MIP-1alpha. Similar degrees of protection were seen for the methylating agent streptozotocin, but no protection was detected for the chloroethylating agents carmustine or mitozolomide in the samples for which there was protection against the toxic effects of Tz. Whereas the mechanism of this effect remains to be established, the results may have potential immediate clinical application in the attenuation of hematological toxicity after administration of methylating antitumor agents.     

Toxicity of trimetrexate on hemopoietic progenitor cells in normal mice

Single increasing doses of methotrexate (MTX) and trimetrexate (TMQ) were administered to normal mice. Survival of hemopoietic progenitor cells assayed as CFU-S and GM-CFC was determined 24 hr after drug injection. The survival of each population in TMQ-treated animals was not statistically different from that observed in mice treated with MTX. No difference was observed in time-survival curves of hemopoietic progenitor cells comparing TMQ to MTX. TMQ toxicity at the hematological level thus seems comparable to that of MTX.     

The sensitivity to hyperthermia of human granulocyte/macrophage progenitor cells (CFU-GM) derived from blood or marrow of normal subjects and patients with chronic granulocytic leukaemia.

To compare the relative heat sensitivities of human normal and neoplastic cells of the same tissue type, a study was carried out of the relative sensitivities to heat of granulocyte/macrophage progenitor cells (CFU-GM) derived from the peripheral blood and bone marrow of normal subjects and patients with chronic granulocytic leukaemia (CGL). Nucleated haemopoietic cells were incubated at temperatures in the range 41.5 degrees C to 44.0 degrees C for various periods before culture in agar. The results of these experiments showed that CFU-GM from normal blood were consistently less sensitive to damage by heat than normal marrow CFU-GM. There was no comparable difference in the relative heat sensitivities of CFU-GM from blood and marrow of patients with CGL and no significant difference between the heat sensitivities of CFU-GM derived from marrow from normal individuals and patients with CGL. The observed difference in heat sensitivity of CFU-GM from normal blood and marrow accords with other data suggesting that the two progenitor cell compartments are distinct: the blood CFU-GM may represent a more primitive population of committed progenitor cells. In CGL, CFU-GM in the blood may much more closely resemble those in the marrow. The data provide no support for the hypothesis that malignant cells differ intrinsically from their normal counterparts in respect of sensitivity to damage by hyperthermia.     

Leucovorin antagonizes the effects of trimetrexate on mouse hemopoietic progenitors.

Trimetrexate (2, 4, diamino -5- methyl - 6 [3, 4, 5, trimethoxyanilino) methyl] quinazoline) (TMQ) is a non-classic folate antagonist that is used as an antineoplastic and antipneumocystis agent with promising results. TMQ and methotrexate (MTX) toxicities are comparable. Leucovorin (N-5-formyltetrahydrofolate) (LV) is used to prevent the toxic effects of MTX. In this study the effects of LV on TMQ induced hemopoietic progenitor damage are studied in a murine model. Changes of pluripotent stem cells (colony forming units spleen, CFU-S), granulocyte-macrophage committed progenitors (GM-CFC), erythroid committed progenitor (BFU-E) levels in the bone marrow were followed after administration to mice of a single dose of TMQ or of simultaneous injection of TMQ and LV. Results show that the latter significantly reduces the effects of the former on peripheral blood cells and on hemopoietic progenitors.     

The long-term effects of MVPP chemotherapy for Hodgkin's disease on bone marrow function

Using in vitro techniques, bone marrow (BM) function has been studied in 25 patients in complete remission and at least one year after the completion of MVPP chemotherapy for Hodgkin's disease. The numbers of granulocyte/macrophage (GM-CFC) and fibroblastoid (CFU-F) progenitors were significantly lower than controls and there was no evidence of any improvement with time (median months off treatment was 30 for GM-CFC and 34 for CFU-F). In long-term BM culture production of haemopoietic cells were strikingly lower in the post-MVPP group and the development of adherent stromal cell populations was also significantly less. In addition, the yield of GM-CFC in adherent layers after four weeks of culture was significantly lower than in controls. We conclude that following MVPP chemotherapy and in apparently disease free and haematologically normal individuals there is evidence of impaired BM function up to nine years after the completion of treatment. These abnormalities may be relevant to the known increased risk of acute non-lymphocytic leukaemias in this group of patients and are likely to render the BM less able to withstand subsequent insults such as further chemotherapy or infection. The eventual development of BM failure is also a possibility and long-term follow-up of these patients is essential.     

Hematological effects in dogs after sequential irradiation of the upper and lower part of the body with single myeloablative doses

The compensating mechanisms determining the tolerance of the hemopoietic system to sequential hemibody irradiation (HBI) with large single doses, the regeneration of the irradiated bone marrow and the long-term effects of such treatment were studied in dogs. The main emphasis was laid on the determination of the granulocyte/macrophage progenitor cells (GM-CFC) in the bone marrow and blood. The general pattern of events in the GM-CFC compartment after each exposure was similar. Irradiation with a dose of 11.7 Gy of the upper body (UBI), that involved the abrogation of approximately 70% of the total active marrow, was followed by an immediate increase in the proliferation and differentiation of GM-CFC in the protected bone marrow. Repopulation of the GM-CFC in the irradiated sites most probably due to seeding of hemopoietic cells from the protected marrow already became evident at day 7 after UBI. At day 56 after UBI, when the irradiation of the lower body (LBI) was performed, the GM-CFC had recovered to between 30 and 40% of their pre-treatment values. Despite this incomplete regeneration, the GM-CFC compartment responded to LBI in a similar way as the GM-CFC had in the protected (normal) marrow after UBI, i.e. by an increased proliferation for at least 21 days. Already at day 7, the bone marrow of the iliac crest that had been exposed to LBI showed a considerable number of GM-CFC. Within no more than 370 days all the bone marrow sites irradiated during either the first or the second treatment had regained their normal GM-CFC values.     

Enhancing hemopoietic drug resistance: a rationale for reconsidering the clinical use of mitozolomide

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.     

Effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse

This paper reports the effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse. Cyclophosphamide 0.12 mg/g body weight was injected into the mice once and the observation lasted for 2 weeks. After the injection, peripheral leukocytes were reduced to the lowest level on day 4 and then increased higher than the control on day 7 to 14. The number of nucleated cell in the bone marrow was the lowest at the 48th hour and gradually became normal within two weeks. The pluripotent hemopoietic stem cells--CFU-S (colony forming unit-spleen) were depleted abruptly in 24 hours, then reproliferated exponentially to a peak on day 3, followed by a second decrease and came back to normal level on day 11 to 14. The changes of granulocytic progenitor cell CFU-D (colony forming unit-diffusion chamber) and CFU-C (colony forming unit-culture) were quite similar to that of CFU-S but their proliferation peak was on day 4. The peripheral leukocyte drop was slower and the return to normal was earlier than the hemopoietic cells. So the recovery of leukocyte count does not mean a real reconstruction of hematopoiesis. The bone marrow stroma observed by CFU-F (colony forming unit-fibroblastoid) assay and marrow microcirculation were also damaged and did not recover to normal during the observation. The bone marrow stroma and microcirculation showed a more serious damage.     

The effects of acetaldophosphamide, a novel stable aldophosphamide analogue, on normal human and leukemic progenitor cells in vitro: implications for use in bone marrow purging

Acetaldophosphamide (A-ALD), a novel in vitro active and stable derivative of aldophosphamide, kills human bone marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) independent of the cell cycle. The surviving fraction of GM-CFC is an exponential function of the drug concentration and time of exposure. Variation of marrow light-density cell concentration between 2 x 10(6) and 10 x 10(6)/ml does not significantly influence its GM-CFC toxicity. Marrow depleted of GM-CFC by A-ALD subsequently generates GM-CFC when grown in suspension cultures. During the early period after treatment with A-ALD the number of surviving GM-CFC (size of surviving GM-CFC compartment) does influence the speed of the GM-CFC repopulation in suspension cultures. The importance of the number of surviving GM-CFCs for the growth and maintenance of GM-CFC population in such suspension cultures diminishes with time. No significant differences are observed after 2 wk, indicating that the ancestor stem cell population and its regenerative potential responsible for in vitro hematopoiesis have not been significantly affected by the drug treatment. A-ALD-treated progenitor cells retain their ability to integrate with the previously established marrow stromal cell layer and generate GM-CFC within this layer to an extent comparable to that of untreated marrow cells. The effect of A-ALD on human hematopoiesis is comparable to that of 4-hydroperoxycyclophosphamide. Its advantage over 4-hydroperoxycyclophosphamide is a greater stability in vitro. It has sparing effect on GM-CFC ancestor cells. Its toxicity to myeloid leukemia cell line (KBM-3)-derived clonogeneic cells is higher than to the GM-CFC. It is similar in doxorubicin-sensitive (KBM-3) and -resistant (KBM-3/DOX) leukemic cells. Thus, A-ALD appears to be a promising drug for in vitro purging of bone marrow cells.