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     

Residual haemopoietic damage in the mouse after fractionated gamma-irradiation, down to 0.1 Gy per fraction

Residual damage in haemopoietic progenitor cell populations, spleen and granulocyte-macrophage colony-forming cells (CFU-S and GM-CFC) was detected in mice after 15 daily fractions where the dose per fraction was as low as 0.1 Gy. The injury was dose-dependent and after higher total fractionated doses of 7.5-10 Gy the CFU-S population recovered to about 50% of control between 2 and 12 months after irradiation. Residual damage was also detected in the stroma, in the form of reduced numbers of fibroblastoid colony-forming cells and of CFU-S in ossicles under the kidney capsule. The response to a second course of 15 fractions, given 3 weeks after the end of the first course, was similar and additive to the response to the first course in the short term. However, in the long term, recovery levels were similar after either one or two courses.     

Thermal sensitivity of haemopoietic and stromal progenitor cells in different proliferative states

Stromal progenitor cells (CFU-F) in normal mouse bone marrow were more sensitive to heat at 43 degrees C than haemopoietic progenitor cells (CFU-S and GM-CFC) by a factor of approximately 1.2. In marrow regenerating after 4.5 Gy X-rays, the changes in sensitivity were by less than a factor of 1.4 and the sensitivity of CFU-F changed slightly to become intermediate between that of CFU-S and GM-CFC. A comparison of sensitivities reported in the literature revealed an inexplicable large variation of up to a factor of 6 in the thermal sensitivities of CFU-S and GM-CFC.     

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.     

Modifications of pluripotent stem cell differentiation after Ara-C treatment: Clonal analyses of CFU-S progeny

The nature of the mechanisms controlling CFU-S differentiation is a crucial problem in haematology and, thus far, little is known concerning these phenomena. Work done in our laboratory has shown that the distribution of the histologic cell types represented in spleen colonies (CFU-S) differ depending on whether normal bone marrow or marrow from Ara-C treated mice is injected into the irradiated recipients. As measured by the mean of the absolute number of colonies per spleen, bone marrow from Ara-C treated mice gives more erythroid colonies and fewer granulocytic colonies than do cells from normal bone marrow. We have demonstrated that these modifications are under the control of humoral factors. Two significant questions arise from these observations. First, are the colonies after Ara-C treatment derived from a single multipotential cell rather than from already committed progenitors and, second, is this shift in granulocytic-erythroid representation a reflection of modifications at the CFU-S level introduced by our Ara-C system? To answer these questions, we analysed the progeny of each individual spleen nodule either by reinjecting each colony unit into a secondary recipient or by cloning these cells in methyl cellulose with appropriate stimulating factors. We thus determined the number of retransplantable stem cells, as well as the number of committed precursors present in each spleen nodule. Our results demonstrate that most spleen colonies are transplantable and give rise to secondary colonies. These secondary colonies are of all haematological types, therefore proving that the nodules contain CFU-S and that these CFU-S are pluripotent. All spleen colonies contain GM-CFC, even in the nodules that were histologically erythroid. We thus conclude that modifications in the E/G ratio of spleen colonies after injection of bone marrow from Ara-C treated mice are a reflection of changes in CFU-S differentiation pathways.     

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.     

Dynamics of leukemic and normal stem cells in leukemic RFM mice.

RFM mice spontaneously develop a myelogenous leukemia that is transplantable into nonleukemic RFM mice. On transplantation, hemopoietic stem cells from leukemic mice (L-CFU-S) will seed in the spleen and grow as discrete colonies, as will hemopoietic stem cells from normal mice (N-CFU-S). As the leukemic cells used in these experiments have 39 chromosomes and normal murine cells have 40, it has been possible to estimate the numbers of N-CFU-S and L-CFU-S in RFM mice at weekly intervals after these mice had been given i.v. injections of 10(6) leukemic spleen cells (spleen cells from preterminal leukemic mice). At each study time, splenic weights, peripheral blood counts, and nucleated cell counts and colony forming units (CFU-S) of marrow, spleen, and blood were assayed. The karyotypes of dividing cells from and the histology of the resultant spleen colonies were also studied. Two weeks after the injection of leukemic spleen cells, the number of CFU-S in the marrow had increased to 3 to 10 times normal, that in the spleen to 100 times normal, and that in the blood was markedly increased. Three weeks after injection, the number of CFU-S in the marrow fell from the peak level at 2 weeks, the number in the spleen rose modestly, and the number in the blood continued to be markedly increased. A normal distribution of erythroid, myeloid, and megakaryocytic colonies was obtained from CFU-S assayed 1 week after injection of leukemic spleen cells, but from CFU-S assayed 2 or 3 weeks after injection of leukemic spleen cells, the colonies formed were comprised almost exclusively of myeloid cells. From spleen colonies formed from marrow or spleen cells obtained 1 week after the injection of leukemic spleen cells, all karyotypes contained 40 chromosomes, whereas from spleen colonies formed from marrow or spleen cells obtained 2 or 3 weeks after injection of spleen cells, almost all karyotypes contained 39 chromosomes. In contrast, most of the karyotypes found in spleen colonies formed from the injection of blood cells even 3 weeks after injection of leukemic spleen cells contained 40 chromosomes. All colonies containing cells with 39 chromosomes, leukemic colonies, contained only myeloid cells. We conclude that L-CFU-S differentiate only into the myeloid series. Early in the course of the disease there is an increase in both N-CFU-S and L-CFU-S in the spleen and marrow. As the disease progresses, the numbers of N-CFU-S in both spleen and marrow decline and, during the final week of the illness, the number of L-CFU-S in the marrow declines. The CFU-S in the peripheral blood are predominantly of normal type, even late in the disease when N-CFU-S are rare in the spleen and marrow.     

Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors

To investigate cell proliferation in regenerating spleen, bone marrow of normal and gamma-irradiated donor mice (3 weeks after 5 Gy) was transfused into lethally irradiated recipients. In the donors and in the recipient spleens numbers of CFU-S and progenitor cells were determined. In the irradiated donors the progenitors were at control level after 3 weeks of recovery although CFU-S were still at 50% of control. Recipients of the irradiated marrow received therefore an increased proportion of progenitors. CFU-C appeared to be self-renewing and/or increased in number due to enhanced CFU-S differentiation, but not the erythroid progenitors. CFU-S self-renewal was reduced after 5 Gy. The data suggest that cell differentiation and maturation proceed during early splenic regeneration. The quantity of CFU-C does not necessarily mirror the situation in the stem cell compartment.     

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.     

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.     

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.     

The effect of disulfiram on cyclophosphamide-mediated myeloid toxicity

Summary We have previously shown that disulfiram (DSF) blocks the urotoxicity of cyclophosphamide (CYT) in mice and increases the oncolytic effect of CYT in the L1210 murine leukemia. However, mice treated with CYT and DSF appeared to have longer-lasting neutropenia than animals treated with CYT alone. To determine whether DSF uroprotection of CYT-treated mice was associated with increased myeloid toxicity, we examined the effects of DSF plus CYT treatment on the bone marrow granulocyte/macrophage progenitor cell (GM-CFC). Marrow cellularity and GM-CFC numbers were analyzed at 1, 2 and 3 days after injection of CYT (62.5 or 125 mg/kg) or CYT plus DSF (200 mg/kg). CYT alone caused a decrease in total marrow cellularity varying from 20% to 50% of control. Animals given CYT plus DSF had a somewhat greater decrease in total marrow cellularity than those treated with CYT alone. However, in mice treated with CYT plus DSF, the GM-CFC were relatively well preserved and the recovery of the GM-CFC was not prolonged by DSF. It appears from these studies that the acute toxic effect of CYT on the granulocyte/macrophage progenitor cells is not enhanced by DSF.     

Role of pluripoietins in murine bone marrow stem cell differentiation

The decision of the differentiation pathways taken by pluripotent stem cells seems to be under the influence, at least in part, of humoral factors acting at the CFU-S level. This differentiation is assessed by histological examinations of the recipient spleen colonies in order to determine the E/G ratios. In vitro cultures are made to determine GM-CFC and BFU_E concentrations. After AraC treatment, CFU-S differentiate preferentially towards erythropoiesis. After total body irradiation, preferential differentiation is toward granulopoiesis. In both cases, there is a competitive phenomenon between the two cell lineages. This is observed in vivo and also in the in vivo-in vitro experiments where the responder cell population is in contact only with the eventual diffusible factors and not with the secreting cells or with the drug. These factors do not seem to be EPO, in the case of AraC, nor GM-CSF in the case of irradiation. We therefore suggest that humoral mediators other than those acting at the progenitor cell level can modulate CFU-S differentiation in a specific way after various types of aggression to the bone marrow.     

Suppression of marrow stromal cells and microenvironmental damage following sequential radiation and cyclophosphamide

Persistent defects in marrow stroma may contribute to hemopoietic insufficiency in patients treated with combined modality therapy for malignancy. To assess the bone marrow failure following combined therapy, mice received intraperitoneal administration of four weekly doses of cyclophosphamide, 160 mg/kg (CY) one week after 1500 rad leg irradiation (LI). This treatment inhibited repopulation of endogenous nucleated cells to less than 60% of normal at one, two, four and six months post-irradiation. Leg irradiation alone suppressed the repopulation to about 75% of normal and cyclophosphamide alone suppressed to 80% of normal. Normal body weight and recovery of peripheral leukocytes and hematocrit to normal levels demonstrated the localized nature of the lesion. Differential marrow counts revealed that 1500 rad LI + CY suppressed erythroblasts, myeloid, and lymphoid cells more than either modality alone. To directly assess the damage of sequential 1500 rad LI + CY on the microenvironment, marrow stromal cells were flushed from the femoral marrow and cultured as adherent cell colonies. They were suppressed to less than 30% of normal for three months following combined modality treatment. Other progenitors, granulocyte-macrophage and spleen colony forming units (CFU-C and CFU-S respectively), were suppressed but exhibited different patterns of partial recovery. We conclude that multiple courses of cyclophosphamide starting one week after 1500 rad LI produced persistent damage to the microenvironment reflected by decreased marrow stromal cells and failure of hemopoietic cells and their progenitors to completely repopulate femoral marrow.     

The role of prothymocytes in radiation-induced leukemogenesis in C57BL/Rij mice

Following transplantation of bone marrow into recipient mice subjected to irradiation with four weekly fractions of 1.8 Gy, donor progenitor cells were found to compete with host elements for colonization of the thymus. The capacity for thymus repopulation of bone marrow cells from 4 X 1.8 Gy irradiated mice is decreased to a similar low level as that of regenerating bone marrow derived from lethally irradiated mice reconstituted with isogeneic marrow. It was calculated that the prothymocyte content of both types of regenerating marrow was less than 0.1% of that of normal marrow. Long-term cultures of C57BL bone marrow were found to contain the same numbers of prothymocytes per CFU-S as normal bone marrow, in contrast to cultured marrow from (C3H X AKR)F1 mice, that shows a striking decrease of prothymocytes. Normal bone marrow cells and long-term cultured bone marrow cells of C57BL mice were about equally effective on the basis of numbers of CFU-S injected in protecting 4 X 1.8 Gy irradiated syngeneic recipients from developing thymic lymphomas, while regenerating bone marrow was virtually nonprotective. These data are interpreted as supporting the notion that prothymocytes from the bone marrow play a crucial role in the prevention of radiation-induced thymic lymphomas.     

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.     

Similar effects on murine haemopoietic compartment of low dose rate single dose and high dose rate fractionated total body irradiation. Preliminary results after a unique dose of 750 cGy

This study was designed to compare two different modalities of TBI which are currently used in clinical practice. The same dose of 750 cGy was given to CBA mice either in a single dose at a low dose rate (4 cGy min-1) (STBI) or in a fractionated regimen (six fractions of 125 cGy three times a day) at a higher dose rate (25 cGy min-1) (FTBI). After TBI completion we simultaneously studied the in vivo radiation response of bone marrow cells, two murine bone marrow clonogenic cells (CFU-S and GM-CFC) and peripheral blood lymphocytes and granulocytes for a period of 1 month. The percentage of spleen erythrocytic and granulocytic colonies was also determined. No significant differences were observed between the two groups in the first 48 hours after irradiation except in bone marrow cell numbers, probably due to differences in the overall treatment time between the two TBI schedules. After the first 48 hours the repopulation patterns of the different cells were very similar in both groups. These findings suggest that the different dose rates and fractionation used in this study caused similar radiation damage to the murine haemopoietic system. Moreover, no significant repopulation occurred during the longer overall treatment time of the fractionated regimen. These preliminary results must be corroborated with a larger range of doses before any firm conclusion can be drawn.     

Effects of cisplatin on different haemopoietic progenitor cells in mice.

The effects of Cisplatin on marrow haemopoietic progenitor cells, WBC and RBC were measured and compared in F1 (CBA x C57BL) female mice. Dose/survival curves of Cisplatin for CFU-S, CFU-C and BFU-E were found to be simply exponential, indicating that the effect of the drug has no cell-cycle dependency. BFU-E also appeared significantly (P less than 0.001) more sensitive to Cisplatin than CFU-S and CFU-C. After a single dose of 12 mg/kg of Cisplatin, WBC, MNC and CFU-E were seen to be markedly less reduced and to recover much earlier than CFU-C, and particularly BFU-E and CFU-S. Results suggest that the drug is more toxic for earlier haemopoietic progenitor cells than for the more mature cells, and that the latter are not reliable parameters for complete haemopoietic recovery in mice after treatment with this agent. In the animals treated, there was also a subsequent significant decrease of the RBC count, accompanied by a marked increase of the marrow CFU-E concentration. Possible underlying mechanisms (e.g. alterations of RBC after exposure to Cisplatin) were discussed.     

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.     

Residual injury to the hemopoietic microenvironment following sequential radiation and busulfan

Our earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Because others have shown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a peristent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50 % of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: 1) sequential radiation and busulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoieticrepopulation longer than a similar regimen of cyclophosphamide.