Recovery patterns of rat hemopoietic stem cells between pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA) applied in a leukemogenic regimen.

Male Wistar rats received repeated pulse doses of 7,12-dimethylbenz(a)anthracene (DMBA), known to elicit myelodysplasia followed by acute, mostly erythroblastic, leukemia at 10-day intervals. The recovery of spleen colony forming hemopoietic stem cells (CFU-s) surviving the cytocidal action of DMBA was examined between pulses. Recovery after a pulse of 35 mg/kg body weight varied with the organ source of the CFU-s (femoral bone marrow or spleen) and the number of preceding DMBA pulses. After a single DMBA pulse bone marrow CFU-s initially recovered faster than reported for normal bone marrow CFU-s transplanted into chemically conditioned rats. But recovery was followed by regeneration arrest. Population doubling times of marrow CFU-s increased with the number of DMBA pulses. Recovery of splenic CFU-s was slower after a single DMBA pulse than reported for normal spleen CFU-s transplanted into chemically conditioned rats. The CFU-s population doubling times were not significantly different after a single or five DMBA pulses. After three pulses, however, recovery of splenic CFU-s was exceedingly slow until day 5 and subsequently accelerated, but was still slower than after one or five pulses. In the spleen CFU-s recovery was always accompanied by regeneration of total cell numbers with a preference for erythroid regeneration. In the bone marrow this was the case after three DMBA pulses only.

The possibility of assaying Wistar rat bone marrow CFUs in a xenogeneic (rat-to-mouse) system.

The possibility of replacing the space-consuming rat-to-rat colony-forming unit (CFUs) assay by rat-to-mouse assay systems was examined using Wistar rat bone marrow. After considering the published results on the responsiveness of mouse strains to hemopoietic xenografts and on the ways to abrogate "xenogeneic resistance', we tested C57B1/6J and C3H/He mice conditioned by cyclophosphamide (CY) and/or whole-body irradiation in the following combinations: 850 rad C57Bl; 850 rad + CY C57Bl; 800 rad + CY C3H. A linear relationship between the number of cells injected and the macroscopical spleen colony count could be demonstrated with all three combinations. However, we observed a high number of endogenous colonies in the 850 rad C57B1 system. The results were confirmed by karyotype analysis. Colony yield and seeding efficiency with 800 rad + CY C3H were comparable to the rat-to-rat assay, but were considerably lower in the case of 850 rad + CY C57B1. In the latter system, the colonies were primarily erythroid.

S-phase resistance of rat hematopoietic stem cells to 7,12-dimethylbenz(a)anthracene cytocide and its implications for leukemia development.

In the experimental rat leukemia system, induced by repeated 7,12-dimethylbenz(a)anthracene (DMBA) pulses, the sensitivity of the spleen colony-forming hematopoietic stem cells (CFU-s) to the cytocidal action of a challenging DMBA injection (35 mg/kg body weight) varied with the number of pulses already applied and the organ source of CFU-s (femoral bone marrow or spleen). Assessment of the fraction of DNA-synthesizing CFU-s with the [3H]thymidine suicide technique at the time of DMBA challenge and comparison with the 20-hr CFU-s reduction values by DMBA in vivo showed an inverse correlation (p less than 0.001). It was deduced, therefore, that S-phase CFU-s are relatively resistant to DMBA cytocide. Since initiation by chemical carcinogens has been shown to be relatively S-phase specific, S-phase-resistant cytocide would lead to a selection of initiated cells and, in the case of repeated applications, to a selection of cells with multiple successive initiation hits. Preferential differentiation and organ site of leukemia, as well as evolution in sequential morphological steps, fit this assumption.