Experimental studies of radiation-induced leukaemia.
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Animals↗
Biomedical subjects
E G Wright
Publications and source records attributed to E G Wright.
Oral (po) dosing with RSU 1069 or RB 6145 maintains their potency as hypoxic cell radiosensitizers and cytotoxins but reduces systemic toxicity compared with parenteral (ip) administration in mice.
RB 6145 is a pro-drug of the hypoxic cell radiosensitizer RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for C3H/He mice was 80 mg/kg (0.38 mmol/kg) ip but 320 mg/kg (1.5 mmol/kg) following po administration. The MTD values of RB 6145 were 350 mg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 toward bone marrow stem cells was also less after po administration than after ip administration; 0.1 mmol/kg ip RSU 1069 and 0.38 mmol/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. Oral administration of RSU 1069 resulted in lower spermatogenic toxicity. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Some nephrotoxicity was observed in half of the mice given the highest po dose of 1.5 mmol/kg of RSU 1069; this was not observed following the highest ip dose of drug. For RSU 1069 and RB 6145, administered by either route, the maximum hypoxic cell radiosensitization in murine KHT sarcomas, occurred when the drugs were given 45-60 min before 10 Gy of X rays. The degree of radiosensitization produced by a particular dose of either compound was largely independent of the route of administration. Preliminary pharmacokinetic studies, using 3H-RSU 1069, suggested that anti-tumor efficacy correlated with peak blood level of label and concentration in the tumor at the time of irradiation, which were not reduced by po compared with ip administration. Normal tissue toxicity tended to correlate with total exposure over time, which was reduced approximately two-fold by po administration. Oral administration of RSU 1069 or RB 6145, as well as being convenient, may give therapeutic benefit since dose-limiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was less affected.
Administration, Oral↗
Identification and characterization of an inhibitor of haemopoietic stem cell proliferation.
The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.
Amino Acid Sequence↗
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.
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Lack of acute effects of 20 mT, 50 Hz magnetic fields on murine haemopoiesis.
Some epidemiological studies have drawn attention to a possible association between exposure to extremely low-frequency (ELF) electromagnetic fields and the development of acute myeloid leukaemia (AML) in adults. At present there is no experimental evidence for such an association. We have investigated the acute effects of power frequency magnetic fields on haemopoiesis in CBA/H mice known to be susceptible to the induction of AML after exposure to ionizing radiation. Up to 19 days after exposure to 50 Hz fields at 20 mT for 7 days no significant effects on peripheral blood characteristics were observed. Assays of the bone marrow stem cells and myelomonocytic progenitor cells also failed to reveal significant effects. Our experiments cannot, however, rule out subtle effects on cell population dynamics, and further investigations, including long-term studies, are required to establish the extent to which ELF magnetic fields might affect the haemopoietic system.
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Late effects of X-irradiation on haemopoietic stem cells in CBA/H mice.
CBA/H male mice have been used to study the recovery of haemopoietic stem cells following 3 Gy whole-body X-irradiation. Despite significant proliferation haemopoietic stem cells (measured as spleen colony-forming units or CFU-S) did not recover to control values and their numbers remained sub-optimal at 600 days post-irradiation. At this time there were significant increases in both proliferation and self-renewal characteristics of the CFU-S but no impairment of the capacity of bone marrow cells to rescue and repopulate lethally irradiated transplant recipients.
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Local regulation of haemopoietic stem cell proliferation in mice following irradiation.
Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.
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A novel in vitro assay for murine haematopoietic stem cells.
Study of the biology of haematopoietic stem cells is crucially dependent on the availability of suitable in vitro assays. Existing assays have suffered from the fact that they detect small subcompartments of the total stem cell compartment. This limits experiments where it is required to assay a high proportion of stem cells, e.g. the enumeration of stem cell numbers under varying conditions or the identification and purification of stem cell regulators. We describe an in vitro assay which shows macroscopic colony formation and limited self-renewal capacity in vitro. The detected cell (CFU-A) has a low cycling status in normal bone marrow (NBM) and responds to known stem cell regulators. The incidence (100-200 per 10(5) in NBM), the proliferative characteristics under stress and some of the physical properties are similar to stem cells detected by colony formation after transplantation into lethally irradiated recipients (CFU-S). These data indicate that our assay detects a high proportion of haematopoietic stem cells in vitro. This will facilitate experiments on stem cell behaviour which have previously been difficult to conduct.
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The effect of stem cell proliferation regulators demonstrated with an in vitro assay.
Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.
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The production of factors regulating the proliferation of haemopoietic spleen colony-forming cells by bone marrow macrophages.
Media conditioned by normal murine bone marrow cells contain an inhibitor of haemopoietic spleen colony-forming cell proliferation that is concentrated in a nominal 50-100K fraction. Media conditioned by regenerating marrow cells contain a proliferation-stimulatory activity that is concentrated in a nominal 30-50K fraction. Cell separation experiments demonstrated that the activities are produced by adherent, phagocytic, radioresistant, Thy 1.2- Fc+, F4/80+ cells. Cultured macrophages, obtained from long-term marrow cultures or derived from progenitor cells in methyl cellulose cultures are also capable of producing inhibitory and stimulatory activities. The results are consistent with macrophages being an important source of stem cell proliferation regulators in the bone marrow.
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Haemopoietic stem cell proliferation in the bone marrow of S1/S1d mice.
In marrow from Sl/Sld mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sld marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sld marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sld CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment. Abnormalities in Sl/Sld CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sld donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sld CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sld or +/+ marrow. Sl/Sld mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.
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Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur.
Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.
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Ectopic implantation studies using Sl/Sld marrow and recipients.
Marrow from Sl/Sld mice (in which the hemopoietic stromal microenvironment is defective), when implanted beneath the renal capsule of a normal littermate, produces an ectopic marrow site containing the same number of stem cells (CFU-S) and nearly as many GM-CFC as that obtained by implanting marrow from a normal littermate. On the other hand, a marrow plug from an Sl/Sld donor implanted beneath the renal capsule of an Sl/Sld littermate produces less than half the number of CFU-S and about 10% of the number of GM-CFC. This suggests that the recipient of the ectopic implant can contribute in some way to the stromal environment of the grafted marrow.
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A stimulator of murine haemopoietic stem cell proliferation produced by human fetal liver cells.
Normal murine bone marrow was incubated with medium conditioned by liver cells from human fetuses of 11-17 weeks gestational age. This treatment increased the proportion of murine haemopoietic stem cells (CFU-S) which were synthesizing DNA from less than 10% to more than 30%. The human fetal liver cell supernatants were fractionated using Amicon filters to obtain nominal molecular weight ranges of 10-30,000, 30-50,000 and 50-100,000 daltons. The stimulator was present only in the 30-50,000 dalton fraction. When human fetal liver cells were separated by adherence to plastic, medium conditioned by the adherent, but not the non-adherent, cells produced the stimulator. A population of non-cycling GM-CFC was not switched into cycle during incubation with this CFU-S proliferation stimulator. Human fetal livers of all gestational ages tested contain a specific stimulator of CFU-S proliferation. This appears to have properties similar to that demonstrated in murine fetal liver and regenerating murine bone marrow. Human and murine haemopoietic stem cell proliferation may therefore be regulated by similar mechanisms.
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Haemopoietic stem cell proliferation: spatial and temporal considerations.
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Animals↗
Regulators of stem cell proliferation in the haemopoietic tissues of W/Wv and S1/S1d mice.
The presence of CFU-S proliferation stimulatory and inhibitory activities in the bone marrow and spleens of normal mice (+/+) and mice with mutations affecting the proliferative behaviour of stem cells (S1/S1d and W/Wv) has been investigated. S1/S1d and +/+ bone marrow and spleen contain virtually no detectable stimulator but the corresponding tissues from W/Wv mice are both strongly stimulatory. S1/S1d marrow in particular, but also +/+ marrow are strongly inhibitory whilst +/+ spleen, S1/S1d spleen, W/Wv marrow and W/Wv spleen all contain inhibitory activity but at a lower specific activity. The data are compatible with studies of cell and tissue grafts that have indicated an intrinsic haemopoietic stem cell defect in W/Wv mice and an extrinsic, microenvironmental defect in S1/S1d mice. It is suggested that they are also compatible with defective regulatory interactions between stem cells and regulator-producing cells and that the W and S1 loci may code for products involved in the production of, or response to, CFU-S proliferation regulators.
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Effect of haemopoietic stem-cell proliferation regulators on early and late spleen colony-forming cells.
An inhibitor and stimulator of CFU-s proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-s (e.g. normal bone marrow) and proliferating CFU-s (e.g. regenerating bone marrow). Their effects on the proliferative behaviour of steady-state and regenerating marrow CFU-s, which produce colonies 7, 10 and 12 days post-transplantation have been investigated. The results demonstrate changing sensitivities of CFU-s to inhibitor and stimulator as they progress through a developmental age structure, 'Older' CFU-s (producing early spleen colonies) are more sensitive to stimulator, 'Younger' CFU-s (producing late spleen colonies) are more sensitive to inhibitor.
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