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.

Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang.

Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals. Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang. In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed. We have measured telomere length in different cell types from wild-type and Ku86-deficient mice. In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang. Interestingly, Ku86-/- cells show telomeric fusions with long telomeres (>81 kb) at the fusion point. These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.

Chromosomal aberrations of blood lymphocytes induced in vitro by radon-222 daughter alpha-irradiation.

Blood samples were irradiated in vitro with alpha-rays emitted from short-lived radon decay products dissolved in the culture medium at doses between 0.03 and 41.4 mGy. The data were collected from experiments conducted during the period 1984-1992 and comprise a total of about 64000 scored metaphases. For statistical reasons, only 60,022 metaphases were used for the subsequent analysis. The results for total chromosome aberrations and dicentrics indicate a linear dose dependence in the dose range above about 10 mGy, consistent with other experimental observations. At doses below about 10 mGy, aberration frequencies cannot be linearly extrapolated from higher doses, suggesting that there is no dependence on dose within a certain low-dose range. In addition, a statistically significant minimum has been observed at a dose of about 0.03 mGy, which is consistently lower than the related control values. The behavior of the aberration frequencies in the low-dose region seems to be influenced by the control values, which also depend on the environmental radiation burdens to the donors before blood sampling and thus were significantly affected by the Chernobyl fallout.

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.

The role of the tumor suppressor p53 in spermatogenesis.

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.

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.

Non-random chromosomal involvement in radiation-induced translocations in mouse spermatogonial stem cells.

X-ray induced chromosomal translocations were studied in mouse spermatogonial stem cells by meiotic analysis at the spermatocyte stage many cell generations after induction. Using a composite DNA probe for mouse chromosomes 1, 11 and 13, in combination with fluorescence in situ hybridization, the involvement of these three chromosomes in translocation formation was recorded. The obtained results at various sampling times ranging from 75 to 320 days after irradiation show no significant differences in the participation pattern of the painted chromosomes in multivalent formation with increasing sampling time. Pooling of the data at the different dose levels of 5 and 7 Gy indicated significant overrepresentation of the specifically stained bivalents in translocation formation. This suggests that clonal proliferation cannot be held responsible for the observed non-randomness. Experiments with the DNA-repair inhibitor 3-aminobenzamide showed no change in the recorded overrepresentation, indicating that it is probably not the repair of lesions that is causing this phenomenon but rather non-random induction.

Comet assay demonstrates a higher ultraviolet B sensitivity to DNA damage in dysplastic nevus cells than in common melanocytic nevus cells and foreskin melanocytes.

We used the single cell gel electrophoresis assay (comet assay) to study ultraviolet B (UVB)-induced DNA damage in pigment cells. This assay detects DNA damage, mainly DNA strand breaks and alkali labile sites in the DNA molecule. We studied the effect of biologically relevant doses (comparable to 2-3 MED (minimal erythemal dose) for in vivo irradiated full-thickness skin) of monochromatic UVB light of 302 nm on cultured melanocytes derived from foreskin, common melanocytic nevi, and dysplastic nevi. We were able to demonstrate a linear dose-response relationship between UV dose and the migration coefficient of the comet tail in all three types of pigment cells. Nevus cells originating from dysplastic nevi showed the highest sensitivity to UVB irradiation: 65% higher induction of DNA damage compared to the induction in foreskin melanocytes. Common melanocytic nevus cells were most resistant and showed a 30% lower induction of DNA damage in comparison to foreskin melanocytes. Differences in chromatin structure and cell cycle profile may influence the results of the comet assay. Control experiments with x-ray irradiation, which is well known to produce direct DNA strand breaks via radical formation, revealed only small differences between the three types of melanocytic cells. It is unlikely, therefore, that intrinsic nuclear characteristics may account for the observed differences.

Mitotic and meiotic detection of radiation-induced translocations in mouse stem cell spermatogonia using fluorescence in situ hybridization.

The efficiency of two methods of detection of translocations induced in mouse stem cell spermatogonia by X-ray doses of 2, 5 and 7 Gy was compared: classical multivalent analysis at diakinesis-metaphase I of meiosis and observation via fluorescence in situ hybridization analysis of mitotic or meiotic stages. Specific DNA libraries for chromosomes 1, 11 and 13 were used. The results obtained indicate that (a) chromosomes 1, 11 and 13 are more involved in multivalent formation than expected on the basis of DNA content and (b) if the mitotic FISH analysis data are corrected for the observed over-representation, the frequencies of induced translocations are similar to those recorded in the classical multivalent studies, suggesting equal scoring efficiencies in both systems.

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.

Radioprotective effects of prostaglandins for chromosomal aberrations and cell killing in V79 Chinese hamster cells grown as spheroids in vitro and for mouse spermatogonial stem cells and bone marrow cells in vivo.

The radioprotective effects of prostaglandins (PGE2, PGE1 and its analogue misoprostol (MP) were investigated in cultures of V79 Chinese hamster (CHO) cells grown as spheroids and as monolayers, CHO cells grown as monolayers, and in bone marrow polychromatic erythrocytes and spermatogonial stem cells in mouse. The X-ray doses were 0.75 Gy (hamster cells) and 5, 8 and 10 Gy (mouse experiments). Prostaglandin pre-irradiation treatment resulted in a marked reduction in the frequencies of chromosomal aberrations in V79 spheroids and of reciprocal translocations in mouse stem cell spermatogonia. The amount of mouse spermatogonial stem cell killing was likewise significantly reduced. No radioprotective effects of prostaglandins could be demonstrated, however, for chromosomal aberrations in hamster cells grown as monolayers, for survival of V79 cells grown as spheroids, and for the induction of micronuclei in bone marrow polychromatic erythrocytes of mouse.

The induction of chromosomal damage and cell killing in mouse spermatogonial stem cells following combined treatments with hydroxyurea, 3-aminobenzamide and X-rays.

To analyze in more detail the relation between the sensitivity of spermatogonial stem cells to killing and the induction of genetic damage, mature male mice received combined treatments with hydroxyurea (HU), 3-amino-benzamide (3-AB) and X-rays. Stem cell killing was determined using the repopulation index method and translocations were studied via spermatocyte analysis. HU was administered at 16 or at 48 h before further treatment in order to create stem cell populations with different sensitivities in which the translocation induction and stem cell killing could be studied and compared. The sensitivities for cell death and genetic damage appeared to be strongly correlated: at 16 h after HU significantly higher values were found than at 48 h or in controls without HU pretreatment. By using 3-AB in the treatment schedules we were able to investigate whether the sensitization of stem cells towards cell death and genetic damage is the outcome of a radiation- or drug-induced G1 delay. The effect of 3-AB was most pronounced at 16 h after HU. This confirms that at this interval a large fraction of stem cells is in G1. Our data therefore indicate that all treatments that induce an enrichment of G1 cells also result in a sensitization of stem cells to cell killing or the induction of mutagenic damage.

Reduced recovery of translocations from X-irradiated spermatogonial stem cells of dominant spotting (Wv/+) and steel (Slcon/Slcon) mice.

Dose-response relationships for X-ray-induced reciprocal translocations in spermatogonial stem cells of mutant and wild-type mice were established by spermatocyte analysis many cell generations after irradiation. The mutants studied were Wv/+, the viable allele of dominant spotting in the heterozygous state, and Slcon/Slcon, the homozygous contrasted allele of steel. The results show that the recovered yield of translocations was lowered in both mutants with steel being most extreme. Remarkably, however, no indications for enhanced cell killing were obtained in the mutants, and consequently the peak yields of translocations occurred at about the same dose level (6 Gy) as in normal mice. Histological analysis suggested that the postirradiation recovery of the germinal epithelium was retarded in the mutants with the effect in the steel mice again being most extreme. These differences in differentiation-multiplication patterns of regenerating spermatogonia after irradiation are probably responsible for the reduced recovery of translocations from the mutant mice.

Absence of translocation induction in mouse stem cell spermatogonia by chemical mutagens probably due to selective elimination.

In order to discriminate between different mechanisms put forward to explain the low observed yields of reciprocal translocation configurations at diakinesis-metaphase I spermatocytes following treatment of mouse stem cell spermatogonia with chemical clastogens, the physiological states of stem cells at the moment of treatment were modified. The cell cycle was shortened by pretreatment with 2 Gy of X-rays and/or the DNA repair capacity inhibited by pretreatment with 3-aminobenzamide. Using these conditions the induction of translocations by ethyl methanesulfonate, N-ethyl-N-nitrosourea, methyl methanesulfonate and Adriamycin was studied. The results indicate that selective elimination of aberration carrying cells is probably to a large extent responsible for the low induction rate of translocations in stem cells by chemical clastogens. The high ratio of simple chromosome breaks versus chromosome exchange type aberrations induced by chemical mutagens might be the main cause of the selective elimination.

Meiotic delay of mouse spermatocytes carrying x-ray-induced translocations.

The kinetics of spermatocyte progression through meiotic prophase in cells with or without induced translocations were studied in mice that had been exposed to x-rays. Pulse-labeling experiments using 3H-thymidine, followed by autoradiographic analysis, indicated that at higher x-ray doses (6 and 7 Gy), translocation-carrying cells tend to spend more time in meiotic prophase than do normal cells. At 2 Gy, no such delay seemed to be present. The observed delay may explain the reduction in transmission of translocations to the next generation reported by others.