Role of cell cycle control in radiosensitization of mouse spermatogonial stem cells.

PURPOSE: To investigate the possible role of cell cycle arrest in the radiosensitization of mouse spermatogonial stem cells due to small conditioning X-ray exposures. MATERIALS AND METHODS: A 24 h fractionation interval between conditioning (1 Gy) and challenging (8 or 9 Gy) exposures was used. Two approaches were followed: the first in the Swiss random-bred wild-type mouse of the radiation-induced cell cycle arrest-evading agents 3-aminobenzamide (3-AB) and caffeine; and, second, using the C57BL/6 mouse of different p53 status. As biological parameter stem cell survival was analysed by the repopulation index (RI) method and chromosomal translocations were recorded using spermatocyte analysis at appropriate posttreatment periods. RESULTS: In the Swiss wild-type mouse, the application of 3-AB or caffeine significantly suppressed the sensitization of stem cells towards killing or translocation induction. In the C57BL/6 mouse, somewhat more variability in response was observed but no significant differences in sensitization between the p53 +/+, +/- or -/- mouse were recorded, suggesting no involvement of p53 in this process. CONCLUSIONS: The results indicate that p53-independent cell cycle regulation plays an important role in the radiosensitization of mouse spermatogonial stem cells.

Quantitative correlation between radiation-induced mutagenesis in endogenous genes and transgenes of mouse spermatogonial stem cells.

In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)-X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 +/- 4.4 x 10(-5) was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 +/- 36.1 x 10(-5) was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung.

In vivo and in vitro radioprotective effects of the prostaglandin E1 analogue misoprostol in DNA repair-proficient and -deficient rodent cell systems.

The radioprotective effect of a stable prostaglandin E(1) analogue, misoprostol, was studied in cells from mice with severe combined immunodeficiency (SCID) and in normal cells using X-ray-induced chromosomal aberrations and/or cell killing as the end points. The results clearly show misoprostol-induced radioprotective effects in spermatocytes of the first meiotic division when analyzed for X-ray-induced chromosomal aberrations. The protective effect was independent of Trp53 (formerly known as p53) status. Since spermatocytes are relatively easy to isolate, this appears to be a suitable in vivo model that will allow biochemical studies of the mechanisms involved in radioprotection mediated by misoprostol. Using transfected CHO-K1 cells that stably express a PGE(2) receptor (CPE cells), significant radioprotection mediated by misoprostol from both chromosome breakage and cell death could be demonstrated under in vitro conditions. In addition, evidence was obtained indicating that the degree of radioprotection was dependent on the cell cycle and that S-phase cells were less responsive to misoprostol-mediated radioprotection. These results suggest that CPE cells may be a suitable in vitro model for further studies on the cellular pathways involved in radioprotection by misoprostol in particular and prostaglandins in general.

Further characterization of the radiosensitivity of the scid mouse.

PURPOSE: To further characterize the radiation response of the scid mutation. MATERIALS AND METHODS: X-ray induced chromosomal aberrations and cell killing were analysed using various in vivo or in vitro cell systems. RESULTS: Using low LET X-irradiation a reverse dose-rate effect was found for killing of differentiated and differentiating spermatogonia and the chromosomal hyperradiosensitivity of scid mice was extended to the meiotic prophase. Most striking was the observation made in vitro with synchronized established cell lines that, contrary to the situation in wild-type cells, scid cells display high levels of both chromatid- and chromosome type aberrations when irradiated during the G1-phase of the cell cycle. A time-course for induction of micronucleated polychromatic erythrocytes (MPCE) was determined for scid mice using flow analysis. No significant differences with wild-type mice were recorded. The chromosomal radiosensitivity at the G1 stage in scid cells was 4.3 times higher than in control CB-17 cells whereas G2 sensitivity differed only by a factor of 1.3. CONCLUSIONS: The reportedly normal radiosensitivity for MPCE in scid mice together with previous findings of hypo- or normal radiation sensitivity of scid cells could be explained by the induction of highly lethal chromatid-type damage at the G1 stage of the cell cycle leading to selective elimination of aberration-carrying cells. The differences in chromosomal radiosensitivity between wild-type and scid for the G1 and G2 stage of the cell cycle correlate with variation in the rates of DNA double-strand break (dsb) repair in scid cells during the cell cycle found by others.

Radioprotective effect of misoprostol on mouse spermatogonial stem cells.

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH x 101/F1 hybrid mice (3H1) were analysed by establishing dose--response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1.7 and 3.6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3.6 to 5.0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose--response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.

Differential radioprotective effects of misoprostol in DNA repair-proficient and -deficient or radiosensitive cell systems.

The protective effects of misoprostol (MP), an analogue of prostaglandin E1, on X-ray-induced chromosomal aberrations, were studied in normal or mutant Chinese hamster cell lines grown as spheroids in vitro and on cell-killing in stem-cell spermatogonia of a mutant (acid) mouse strain or its wild-type. The mutant hamster cell lines chosen for this purpose are known to be either hypersensitive to the killing effects of X-rays and/or deficient in the repair of DNA double-strand breaks. The scid mice are deficient in the repair of DNA double-strand breaks. The results show that MP manifests varying degrees of radioprotection in all these systems, but the magnitude of these effects in the mutants is markedly reduced compared to their respective wild-type counterparts. These findings suggest a link between ionizing radiation sensitivity, DNA double-strand break repair capability and MP-mediated radioprotection.

X-ray induced translocations in bone marrow cells of scid and wild type mice detected by fluorescence in situ hybridization using mouse chromosome specific DNA libraries.

Radiation induced chromosomal aberrations in bone marrow cells of scid and normal mice were studied at different sampling times. Fluorescence in situ hybridization (FISH) with DNA libraries specific for chromosomes 1, 11 and 13 was applied to identify the stable types of chromosomal aberrations in addition to the unstable ones. The results obtained confirm earlier observations on stem cell spermatogonia in that, contrary to the situation in normal mice, only very low levels of translocations could be recovered from scid mice at relatively long sampling times (3 weeks). However, studies at a 24 h sampling period demonstrated substantial induction of translocations in scid mice. This suggests enhanced elimination of translocation carrying cells in scid mice during successive cell proliferation, possibly via falling apart of the translocation at the original points of exchange or due to lethal damage at the translocation break points.

X-ray-induced chromosomal aberrations and cell killing in somatic and germ cells of the scid mouse.

To characterize further the radiosensitivity of severe combined immunodeficiency (scid) mice, the induction of micronuclei (MN) in polychromatic erythrocytes as well as cell killing and translocation induction in stem cell spermatogonia was studied. Scid mice turned out to be clearly hypersensitive for X-ray-induced cell killing of both bonemarrow cells and spermatogonial stem cells. The frequencies of recorded micronuclei in polychromatic erythrocytes were comparable with that reported for the normal mouse, whereas the recovery of translocations was extremely low in the scid mouse. The dose-response relationship for induced translocations was bell shaped with a maximum of about 0.5% around doses of 0.5-1.5 Gy X-rays.