Search

The study of substances that inhibit cell proliferation is hampered by the fact that a great many substances will inhibit proliferation in a non-specific way, simply by virtue of their toxic effects. A new assay has been developed, however, to study the proliferation of primitive stem cells in bone marrow in tissue culture dishes. Stem cells are primitive cells that can reproduce and give rise to a variety of different descendent cells. Using this assay, a substance initially called SCI, or stem cell inhibitor, was identified. As the biochemical analysis of SCI continued, it became clear that SCI was identical with the macrophage inflammatory protein-1 alpha, or MIP-1 alpha. This substance is a highly specific inhibitor of stem cell proliferation, and has no effect on the replication of blood progenitor cells, which have already become committed to their adult role. Electrophoresis revealed that the molecule is about 8,000 daltons in molecular weight, but the active substance in tissue culture may exist in complexes that have higher molecular weight. Molecular cloning of the gene for MIP-1 alpha also resulted in the unexpected finding of a closely related substance, known as MIP-1 beta. This substance has no apparent effect on the stem cell colony-forming assay that was used to identify MIP-1 alpha. These findings demonstrate not only that the effect observed with MIP-1 alpha is highly specific, but also that these molecules may be a part of a larger family of molecules, which play a role in the regulation of hematopoiesis, or blood cell production. The inhibition of proliferating stem cells by MIP-1 alpha may have direct clinical importance. Since chemotherapeutic agents kill proliferating cells, whether they are normal or cancerous, the hematopoietic cells are often the first to take a beating during the treatment of cancer patients. Indeed, the toxicity of chemotherapeutic agents for these cells is one of the factors which limits the dosage of chemotherapy. If MIP-1 alpha could be used to temporarily inhibit stem cell proliferation, these cells would be removed from the list of potential targets for chemotherapy; not only would a serious side effect be reduced, but higher and possibly more effective doses of chemotherapy might then be administered. (Consumer Summary produced by Reliance Medical Information, Inc.)

CBA mouse strains have been used for many years as a model of radiation-induced acute myeloid leukaemia and the leukaemias in CBA and their F1 hybrids are characterised by a specific loss of heterozygosity involving one homologue of chromosome 2. Previous cytogenetic studies of transplanted irradiated bone marrow, or of bone marrow obtained from irradiated mice significantly before the appearance of leukaemia, have been interpreted as the chromosome 2 deletion being a high frequency, possibly initiating event. However, these studies had not specifically addressed the question of whether the characteristic deletion was induced at a high frequency in stem cells. Using a PCR-based technique, we have studied the induction of chromosome 2 LOH in the progeny of (CBA/H x C57BL/6)F1 stem cells after a potentially leukaemogenic radiation exposure. Whilst chromosome 2 LOH can be induced directly by irradiation and there is a preferential loss of the CBA allele, the frequency is no greater than LOH induced in other chromosomal regions studied. The data do not support radiation-induced deletion involving one homologue of chromosome 2 in long-term repopulating stem cells (<1 in 200) being as high a frequency event as might be inferred by previous cytogenetic studies of total bone marrow. [ABSTRACT FROM AUTHOR]

Alpha-particle-induced chromosomal instability in haemopoietic cells obtained from the CBA/H, DBA/2 and C57BL/6 inbred strains of mouse has been demonstrated at frequencies dependent on genotype. The CBA/H and DBA/2 strains may be regarded as 'sensitive' and the C57BL/6 strain as 'resistant'; resistance was dominant in cells from F1 hybrids. Previously, in cultures where we demonstrated radiation-induced chromosomal instability we also demonstrated an enhanced and persisting oxyradical activity. Quantitative differences in superoxide generation have now been correlated with genetically determined differences in the expression of chromosomal instability. Our findings demonstrate an important influence of genetic factors in alpha -particle-induced chromosomal instability. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of preconception paternal irradiation (PPI) from injected 239Pu on the susceptibility to induction of lympho-haemopoietic malignancy by subsequent irradiation or exposure to a chemical carcinogen. Materials and Methods: The male CBA/H and DBA2 mouse was injected with 0, 128 or 256Bqg -1 239Pu 12 weeks before mating with the normal CBA/H and C57Bl female respectively. CBA/H offspring were exposed to 3.3Gy gamma -rays total body irradiation: BDF offspring were injected with 50mgkg -1methyl nitrosourea (MN1 U). The offspring were assayed for changes in bone marrow progenitor cell numbers and chromosome aberrations and were followed up for subsequent induction of neoplasia. Results: While the untreated mouse showed a normal distribution for cellularity, spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), significant numbers of PPI offspring presented levels outside the normal range. There was a tendency for them also to show increased, dose-related, levels of chromosomal aberrations. Offspring treated with irradiation or MNU developed an increased incidence of lymphohaemopoietic malignancies. Conclusions: These studies have shown that PPI results in offspring that are more susceptible to the induction of lymphohaemopoietic malignancy on encountering a secondary carcinogenic insult. This may be linked to inherited chromosomal instability and abnormal kinetics of haemopoiesis. The experiments indicate a potential mechanism by which an increased incidence of leukaemia may be linked to PPI. [ABSTRACT FROM AUTHOR]

Forty primary leukaemias that arose in vivo as a consequence of 3 Gy X-irradiation of inbred mouse strains were analysed for Y-chromosome aberrations by conventional cytogenetics and fluorescent in situ hybridization (FISH). Compared with control mice which were X-irradiated but which exhibited no overt signs of leukaemia, the loss and gain of Y-chromosomes in leukaemic spleen cells defined subclonal variants in the radiation-induced haempoietic malignancies that arose in CBA/H, DBA/2 and (C57BL/6 DBA/2)F1 mice. This Y-chromosome instability was significantly higher than that observed in spleen cells of age-matched (or older) irradiated control mice that had not developed overt leukaemia. The detection of Y-chromosome aberrations is considered in the context of the high numbers of potential gene regulatory sequences in the murine Y-chromosome and the potential for the insertional activation of cellular genes during multi-stage radiation leukaemogenesis. [ABSTRACT FROM AUTHOR]

Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-beta-galactosidase activity following whole body irradiation. Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes. The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice.

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Using a bone marrow transplantation protocol in which we transplanted a mixture of irradiated and nonirradiated bone marrow cells that were distinguishable by a cytogenetic marker, we have demonstrated chromosomal instability in the progeny of nonirradiated hemopoietic stem cells. This first demonstration of a link between a bystander effect of ionizing radiation and the induction of genomic instability in vivo clearly poses a major challenge to current views of the mechanisms of radiation-induced DNA damage with mechanistic implications for the health consequences of radiation exposure particularly in the context of the induction of malignancy.

We have demonstrated chromosomal instability in the clonal descendants of hemopoietic stem cells after irradiating murine bone marrow with alpha-particles. However, because cells that are irradiated by alpha-particles are defined by a Poisson distribution of individual particle traversals, there is an inevitable proportion of unirradiated cells in the surviving population. The calculated expected proportions of irradiated and nonirradiated cells indicate that the number of clonogenic cells transmitting chromosomal instability is greater than the number expected to be hit and survive. To investigate further this discrepancy, we studied the effects of interposing a grid between the cells and the alpha-particle source so that the surviving population consists predominantly of untraversed stem cells. Comparison with the same irradiation conditions without the grid reveals that the same level of instability is induced. The data confirm that alpha-particles induce chromosomal instability but instability is demonstrated in the progeny of nonirradiated stem cells and must be due to unexpected interactions between irradiated and nonirradiated cells. This untargeted effect has important implications for mechanistic studies of radiation action and for assessment of radiation risk.

Radiation-induced M361 leukemia bearing mice (M361 mice) show characteristics of acute myelogenous leukemia (AML) with granulocytosis. Granulocyte-macrophage colony forming unit (CFU-GM) increased in the bone marrow and spleen and high activity of colony stimulating factor (CSF) was found in the sera of M361 mice. A cell line (HB-1) was established from the spleen cells of M361 mice. Injection of HB-1 cells induced a similar leukemic response as M361 in syngeneic mice. HB-1 cells did not survive in the suspension culture, but proliferated when cultured on hemopoietic supportive stromal cells (MS-10). HB-1 cells appear to be strictly dependent on the hemopoietic supportive bone marrow stroma, which would provide a useful model for the study of cell-cell interactions between hemopoietic cells and marrow stromal cells.

Earlier work in this laboratory identified high levels of non-clonal chromatid aberrations in the clonal descendants of murine hemopoietic stem cells after many cell divisions postirradiation with densely-ionizing, high linear energy transfer (LET) alpha-particles, but not with sparsely ionizing low LET X-rays. Using the Hprt locus as a marker, we have now demonstrated genomic instability in murine hemopoietic stem cells for greater than 20 cell divisions following both high and low LET irradiation. The increase in Hprt-deficient variants demonstrated following X- as well as alpha-particle and neutron irradiation indicates that there is a difference in the LET-dependence of delayed gene mutations and higher-order cytogenetic effects. Over 90% of the mutations identified arose in the expanding colonies after more than 12 cell divisions postirradiation and therefore cannot be attributed to the initial DNA damage. Such a high frequency of delayed mutations has important implications for mechanistic studies of radiation mutagenesis and for risk estimation.

We have previously reported non-clonal cytogenetic aberrations in the clonal descendants of murine haemopoietic stem cells irradiated in vitro with alpha-particles. The data are consistent with a transmissible chromosomal instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later. To determine whether this instability is transmissible in vivo, bone marrow cells obtained from the male mouse have been irradiated with alpha-particles in vitro and transplanted into female recipients. In the repopulated haemopoietic system we have observed persisting chromosomal instability up to 1 year post-transplantation. The findings demonstrate a long-lived effect of the alpha-particle-induced lesion in the donor repopulating stem cells.

alpha-particles, which are ionising radiation of high linear-energy-transfer emitted, for example, from radon or plutonium, pass through tissue as highly structured tracks. Single target cells in the path of the tracks might be damaged by even low-dose alpha-irradiation. We found non-clonal cytogenetic aberrations, characterised by a high frequency of chromatid aberrations with chromosome aberrations, in clonal descendants of haemopoietic stem cells after exposure to alpha-particles of bone marrow cells from two of four haematologically normal individuals (up to 25% abnormal metaphases). The data are consistent with a transmissible genetic instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later.

Inactivation of murine haemopoietic stem cells, assayed as spleen colony-forming units (CFU-S), has been determined after in vitro irradiation under well defined conditions with small numbers (0.15-3) of alpha-particles of incident energy 3.3 MeV (LET 121 ke V microns-1). Exponential survival curves were obtained with inactivation dose coefficients of 1.83 +/- 0.11 and 1.63 +/- 0.06 Gy-1 for 8 and 12-day CFU-S, respectively, corresponding to inactivation cross sections of 35.6 +/- 2.1 and 31.7 +/- 1.2 microns2. The results indicate that these radiosensitive stem cells have a significant probability of surviving the passage of a single alpha-particle track, estimated at 8 and 18% per particle passage, respectively, if the cells have a diameter of 7 microns. However, this estimation is strongly dependent on the assumed diameter. Conversely, it is demonstrated that the cells cannot have mean geometric diameters of less than 6.7 +/- 0.2 and 6.4 +/- 0.1 microns, respectively. Associated experiments have shown that some cells from the irradiated population do survive to form colonies that express delayed non-clonal chromosome aberrations.

When investigating the biological effects of ionizing radiation on the haemopoietic system, a confounding problem lies in possible differences between the biological effects of sparsely ionizing, low linear energy transfer radiation such as X-, beta- or gamma-rays, and densely ionizing, high linear energy transfer radiation such as alpha-particles. To address this problem we have developed novel techniques for studying haemopoietic cells irradiated with environmentally relevant doses of alpha-particles from a plutonium-238 source. Using a clonogenic culture system, cytogenetic aberrations in individual colonies of haemopoietic cells derived from irradiated stem cells have been studied. Exposure to alpha-particles (but not X-rays) produced a high frequency of non-clonal aberrations in the clonal descendants, compatible with alpha-emitters inducing lesions in stem cells that result in the transmission of chromosomal instability to their progeny. Such unexpected instability may have important implications for radiation leukaemogenesis.

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.

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.

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.

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.

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.

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.