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We examined the effects of hair cycle phase on basal cell carcinoma (BCC) tumorigenesis induced by radiation in mice lacking one Patched allele (Ptc1(neo67/+)). Our results show that Ptc1(neo67/+) mouse skin irradiated in early anagen is highly susceptible to tumor induction, as a 3.2-fold incidence of visible BCC-like tumors was observed in anagen-irradiated compared with telogen-irradiated mice. Microscopic nodular BCC-like tumors were also enhanced by irradiation during active hair-follicle growth phases. Interestingly, histologic examination of the tumors revealed a qualitative difference in BCC tumorigenesis depending on hair growth phase at the time of exposure. In fact, in addition to typical BCC-like tumors, we observed development of a distinct basal cell tumor subtype characterized by anti-cytokeratin 14 and anti-smooth muscle actin reactivity. These tumors showed relatively short latency and rapid growth and were strictly dependent on age at irradiation, as they occurred only in mice irradiated in early anagen phase. Examination of anatomic and immunohistochemical relationships revealed a close relation of these tumors with the follicular outer root sheath of anagen skin. In contrast, there are strong indications for the derivation of typical, smooth muscle actin-negative BCC-like tumors from cell progenitors of interfollicular epidermis. These results underscore the role of follicular bulge stem cells and their progeny with high self-renewal capacity in the formation of basal cell tumors and contribute to clarify the relationship between target cell and tumor phenotype in BCC tumorigenesis induced by radiation.

Hemizygous Ptc1 mice have many features of Gorlin syndrome, including predisposition to medulloblastoma development. Ionizing radiation synergize with Ptc1 mutation to induce medulloblastoma only in neonatally exposed mice. To explore the mechanisms underlying age-dependent susceptibility, we irradiated Ptc(neo67/+) mice at postnatal day 1 (P1) or 10 (P10). We observed a dramatic difference in medulloblastoma incidence, which ranged from 81% in the cerebellum irradiated at P1 to 3% in the cerebellum irradiated at P10. A striking difference was also detected in the frequency of cerebellar preneoplastic lesions (100 versus 14%). Our data also show significantly lower induction of apoptosis in the cerebellum of medulloblastoma-susceptible (P1) compared to -resistant (P10) mice, strongly suggesting that medulloblastoma formation in Ptc1 mutants may be associated with resistance to radiation-induced cell killing. Furthermore, in marked contrast with P10 mice, cerebellum at P1 displays substantially increased activation of the cell survival-promoting Akt/Pkb protein, and markedly decreased p53 levels in response to radiation-induced genotoxic stress. Overall, these results show that developing cerebellar granule neuron precursors' (CGNPs) radiosensitivity to radiation-induced cell death increases with progressing development and inversely correlates with their ability to neoplastically transform., (Oncogene (2006) 25, 1165-1173. doi:10.1038/sj.onc.1209032; published online 9 January 2006.)

We described previously a basal cell carcinoma (BCC) and medulloblastoma (MB) phenotype for CD1Ptch1(neo67/+) mice exposed to ionizing radiation. Ptch1 heterozygous mice mimic the predisposition to BCC and MB development of patients affected by nevoid BCC syndrome that inherit a mutant Patched (Ptch1) allele. To examine the impact of genetic background on development of BCCs and other tumors we used two outbred mouse lines characterized by extremely high, carcinogenesis-susceptible (Car-S), and low, carcinogenesis-resistant (Car-R), susceptibility to skin carcinogenesis. Crosses between Ptch1(neo67/+) mice and Car-S (F1S) or Car-R mice (F1R) were exposed to ionizing radiation. F1SPtch1(neo67/+) mice were highly susceptible to radiation-induced BCCs, whereas F1RPtch1(neo67/+) mice were completely resistant, indicating that tumor penetrance can be modulated by genetic background. Development of microscopic and macroscopic BCC lesions was influenced by Car-S and Car-R genotypes, suggesting a genetic-background effect on both initiation and progression of BCC. Susceptibility was additionally increased in N2 backcross mice (Car-S x F1SPtch1(neo67/+)), showing a contribution from recessive-acting Car-S modifiers. The modifying effects of Car-S-derived susceptibility alleles were tissue specific. In fact, despite higher susceptibility to BCC induction, Car-S-derived lines had lower MB incidence compared with CD1Ptch1(neo67/+) mice. BCC-associated somatic events were not influenced by genetic background, as shown by similar rate of wild-type Ptch1 loss in BCCs from F1SPtch1(neo67/+) (93%) and CD1Ptch1(neo67/+) mice (100%). Finally, microsatellite analysis of BCCs showed Ptch1 loss through interstitial deletion. These results are relevant to humans, in which BCC is the commonest malignancy, because this model system may be used to study genes modifying BCC development.

Loss-of-function mutations in Patched (Ptch1) are implicated in constitutive activation of the Sonic hedgehog pathway in human basal cell carcinomas (BCCs), and inherited Ptch1 mutations underlie basal cell nevus syndrome in which a typical feature is multiple BCC occurring with greater incidence in portals of radiotherapy. Mice in which one copy of Ptch1 is inactivated show increased susceptibility to spontaneous tumor development and hypersensitivity to radiation-induced tumorigenesis, providing an ideal in vivo model to study the typical pathologies associated with basal cell nevus syndrome. We therefore examined BCC development in control and irradiated Ptch1(neo67/+) mice. We show that unirradiated mice develop putative BCC precursor lesions, i.e., basaloid hyperproliferation areas arising from both follicular and interfollicular epithelium, and that these lesions progress to nodular and infiltrative BCCs only in irradiated mice. Data of BCC incidence, multiplicity, and latency support the notion of epidermal hyperproliferations, nodular and infiltrative BCC-like tumors representing different stages of tumor development. This is additionally supported by the pattern of p53 protein expression observed in BCC subtypes and by the finding of retention of the normal remaining Ptch1 allele in all nodular, circumscribed BCCs analyzed compared with its constant loss in infiltrative BCCs. Our data suggest chronological tumor progression from basaloid hyperproliferations to nodular and then infiltrative BCC occurring in a stepwise fashion through the accumulation of sequential genetic alterations.

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D(high-LET) < 5 cGy and D(low-LET) < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.

Individuals affected with the Gorlin syndrome inherit a germ-line mutation of the patched (Ptc1) developmental gene and, analogously to Ptc1 heterozygous mice, show an increased susceptibility to spontaneous tumor development. Human and mouse Ptc1 heterozygotes (Ptc1(+/-)) are also hypersensitive to ionizing radiation (IR)-induced tumorigenesis in terms of basal cell carcinoma (BCC) induction. We have analysed the involvement of Ptc1 in the tumorigenic response to a single dose of 3 Gy X-rays in neonatal and adult Ptc1 heterozygous and wild type mice. We report that irradiation dramatically increased the incidence of medulloblastoma development (51%) over the spontaneous rate (7%) in neonatal but not adult Ptc1 heterozygotes, indicating that medulloblastoma induction by IR is subjected to temporal restriction. Analysis of Ptc1 allele status in the tumors revealed loss of the wild type allele in 17 of 18 medulloblastomas from irradiated mice and in two of three spontaneous medulloblastomas. To our knowledge, irradiated newborn Ptc1(+/-) heterozygous mice constitute the first mouse model of IR-induced medulloblastoma tumorigenesis, providing a useful tool to elucidate the molecular basis of medulloblastoma development.

Outbred carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mouse lines were generated by phenotypic selection for resistance or susceptibility to two-stage skin carcinogenesis. These two Car mouse lines differ by >100-fold in susceptibility. In the present study, we tested the hypothesis that a subset of genetic loci responsible for susceptibility or resistance to chemical skin tumorigenesis may also be involved in radiation-induced skin tumorigenesis. Skin tumorigenesis was tested in groups of Car-S/R mice after X-ray initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. We found that ionizing radiation can initiate skin tumors in Car-S mice but not in Car-R mice. In Car-S mice, the most effective radiation doses (6 and 10 Gy given in four fractions) gave a threefold increase in tumor multiplicity and a twofold increase in tumor incidence compared to a TPA-only control group. We performed a molecular analysis of Hras gene mutations in skin tumors of Car-S mice induced by X-ray initiation/TPA promotion or by TPA promotion alone. The most notable difference emerging from the comparison of these mutation patterns is the high incidence ( approximately 50%) of papillomas lacking Hras gene mutations in X-ray-initiated/TPA-promoted papillomas compared to 13% in papillomas induced by TPA alone, suggesting that lack of Hras gene mutations is a consistent feature of radiation-induced papillomas.

Chromosome 2 (chr 2) deletions are recurrent abnormalities in acute myeloid leukemia (AML) induced by ionizing radiation in the mouse. The localization of deletion sites has proven extremely useful in providing information on the molecular mechanisms of leukemogenesis. The models available for the study of AML are mostly represented by inbred mouse strains, in which the molecular resolution of breakpoints is problematic. In this study, we have examined five leukemic cell lines exhibiting hemizygous chr 2 loss, derived from CBA, C3H, or (C57BLxCBA/H) F1 mice in which AML had been induced by a whole-body dose of radiation. By application of a somatic cell hybridization technique, we have generated interspecific cell hybrids retaining the deleted murine chr 2 homologue. This strategy permitted a very detailed genetic analysis allowing the utilization of any genetic marker on chr 2 without a requirement for polymorphism. Somatic cell hybrid clones were subjected to a high-density polymerase chain reaction-based microsatellite screening using 62-106 informative markers for each cell line. Detailed maps accurately defining chr 2 breakpoints were obtained. The identification of critical breakpoint markers allowed the construction of partial yeast artificial chromosome contigs across chr 2 breakpoints. These maps represent an essential resource for cloning of the breakpoint regions.

An experimental study of male and female CBA/Cne mice was set up at Casaccia primarily to investigate the influence of sex on long-term survival and tumor induction after exposure to high- and low-LET radiation. Mice were whole-body-irradiated at 3 months of age with fission-neutron doses of 0.1, 0.2, 0.4, 0.8, 1.2 and 1.8 Gy at the RSV-TAPIRO reactor (mean neutron energy 0.4 MeV, in terms of kerma, y(D) = 51.5 keV/micron), or with 250 kVp X-ray doses of 1, 3, 5 and 7 Gy. Control and irradiated animals were then followed for their entire life span. As a general finding, male CBA/Cne mice appear more susceptible to tumorigenesis than females. In particular, the incidences of induced acute myeloid leukemia and malignant lymphomas are significant only in male mice. Benign and malignant solid tumors of many types are observed in mice of both sexes, the most frequent being in the lung, liver and ovary. However, evidence for a radiation response is limited to the case of Harderian gland neoplasms. In addition, a comparison of the observed frequency of all irradiated compared to unirradiated animals bearing solid tumors shows that the total tumor occurrence is not altered markedly by radiation exposure. A decrease in survival time is observed for both sexes and radiation types and correlates well with increasing dose. Moreover, both sex and radiation quality appear to influence the life shortening. A similar dose dependence of survival time is found when tumor-free animals alone are considered, suggesting a non-specific component of life-shortening.

An experimental study of the biological effectiveness of multifractionated low doses of high-LET radiation was carried out using BC3F1 male mice. They were treated with whole-body irradiation with five equal daily fractions of fission neutrons to yield cumulative doses of 0.025, 0.05, 0.10, 0.17, 0.25, 0.36, 0.535 and 0.71 Gy at the RSV-TAPIRO reactor (mean neutron energy 0.4 MeV, in terms of kerma, y D = 51.5 keV/microns, dose rate 0.004 Gy/min) and were followed for their entire life span. The statistical method described by Peto et al. (IARC Monograph, Suppl. 2, 1980) to establish the existence of a carcinogenic effect in long-term animal experiments was applied to the data sets. This analysis was done for myeloid leukemia and for the presence of selected solid tumors. Myeloid leukemia was absent in the control group and was rarely found in irradiated animals. However, a positive significant trend was found in the dose ranges 0-0.17 Gy and higher. Epithelial tumors were induced at doses from 0.17 Gy on. Tumor occurrence was evaluated further as final incidences with age adjustment for the differences in mortality rates. Survival and incidence data for selected classes of tumors after 0.17, 0.36 and 0.71 Gy were compared with those from a previous experiment at corresponding doses given acutely (dose rate between 0.05 and 0.25 Gy/min). This indicated no marked overall influence of the time regimen of neutron irradiation on survival and tumor induction.

This paper presents a review of several studies conducted in our laboratory to examine the carcinogenic effects in mice of high-LET radiation and, for comparison, of low-LET reference radiation. For some specific end-points the following conclusions can be formulated: i) the dose-response curves for myeloid leukemia and malignant lymphoma can be interpreted in terms of induction and inactivation; in particular, the data confirmed that a linear dependence of the induction on dose is adequate to describe the response to fission neutrons, while a pure quadratic dependence is consistent with the experimental data for low-LET radiation; ii) in the liver, a marked age-dependence was demonstrated for radiation-induced tumors with a much higher susceptibility in young than in old mice; also for these tumors the dose-effect curves can be described by a linear and a quadratic relationships for high- and low-LET radiation, respectively; iii) data on ovarian tumor induction suggested threshold-like dose responses: these peculiar shapes as well as the absence of a clear radiation quality dependence of the curves are difficult findings to explain using a simple model of radiation action, and they might better be related to a non-stochastic effect of hormonal imbalance following irradiation.

Several experimental studies have been conducted with the objective to improve our knowledge of the types of dose-response relationships for radiation carcinogenesis in mice exposed to single acute doses. The experimental results on tumor induction have already been published and are here summarized with emphasis on the dependence on radiation quality, age at irradiation, and sex. These data indicate that the bone marrow, liver, and ovaries of the mice tested have an appreciable susceptibility to radiation carcinogenesis. However, the shape of the dose-response relationship depends on the tissue exposed. The data also confirm that a linear relationship is adequate for a conservative description of the dose-effect curves after exposure to low dose of neutrons, while a purely quadratic dependence is not inconsistent with the experimental data obtained using low-LET radiation. Other information which stems from the present analysis is that the susceptibility to radiation induction of liver tumor by fission neutrons decreases in old age. Finally, the experimental data on induction of ovarian tumors suggest a threshold-like dose response.

The induction of liver tumors has been studied in BC3F1 male mice after acute whole-body irradiation with fission neutrons and X rays, given at different ages. In prenatally irradiated mice, a small effect is seen after doses of 0.3 to 2.1 Gy of X rays, and a more pronounced effect is found after neutron doses of 0.09 to 0.62 Gy. At 3 months of age the animals show a higher incidence after X-ray doses from 2 Gy, and for neutrons from 0.17 Gy. At 19 months of age, liver tumors are rarely induced by either type of radiation. These findings are confirmed by the statistical analysis of trend. The possibility of deriving dose-response relationships for liver tumors was also investigated. In the dose ranges where the risk appears to increase as a function of the increase in dose, the data points for neutrons were well fitted by a linear expression. A pure quadratic relationship best fitted the X-ray data. Using these expressions, the RBE for neutrons was 28 at 0.09 Gy for prenatal irradiation and 13 at 0.17 Gy for irradiation at 3 months. This suggests the existence of a risk of developing liver tumors after exposure to radiation, and fetal liver seems to be particularly sensitive to neoplastic transformation. This risk may be negligible at low doses (less than 1 Gy) of low-LET radiation, or with exposure at older ages.

The present analysis of data on the induction of lymphoma and myeloid leukemia in BC3F1 mice has indicated some new and interesting aspects regarding the shapes of the dose-effect curves. The incidence data can be interpreted by radiobiological models of the induction process coupled with cell inactivation. In particular, for malignant lymphoma the dose-response curve after X rays can be described assuming a quadratic model corrected for cell inactivation, while the incidence data after fission neutrons are best fitted by a linear model which also allows for cell inactivation. Myeloid leukemia has also been induced in BC3F1 mice. The bell-shaped dose-response curves observed after irradiation with either X rays or neutrons are explained by assuming simultaneous initial transforming events and cell inactivation with the data for cell inactivation at higher doses being in agreement with data reported for other strains of mice. A value for relative biological effectiveness of 4 is obtained at the lowest neutron dose used. The value of the inactivation parameters can be compared with those of the cell inactivation probability per unit dose for the bone marrow hematopoietic stem cells, which are believed to be the target cells for these tumors.

The average final incidence of reticulum cell sarcoma (RCS) in untreated control (C57BL/Cnefemale X C3H/Cnemale)F1 male mice was 57% and was not significantly changed by single acute whole-body doses of X-rays up to 400 rads. Higher doses sharply decreased this incidence to very low levels. In a syngeneic chimera system, however, irradiation (400 rads) of bone marrow prior to transplantation significantly increased the frequency and rate of RCS relative to transplantation of unirradiated marrow. An endogenous repopulating system was tested; marrow was irradiated in situ by a limb-shielding technique. The results indicated a biphasic dose-incidence curve with a peak at 500 rads. The rising part of the curve was in agreement with and extended the conclusion from the exogenous system, whereas the descending portion at higher doses paralleled the trend observed in the whole-body X-irradiated animals. Finally, the irradiation of two hind legs resulted in a higher frequency of RCS than did irradiation of only one leg at the same dose levels.

Extension of previous investigations at this laboratory regarding life shortening and tumor induction in the mouse has provided more complete dose-response information in the low dose region of X rays and neutrons. A complete observation of survival and late pathology has been carried out on over 2000 BC3F1 female mice irradiated with single doses of 1.5 MeV neutrons (0.5, 1, 2, 4, 8, 16 cGy) and, for comparison, of X rays (4, 8, 16, 32, 64, 128, 256 cGy). Data analysis has shown that a significant life shortening is observable only for individual neutron doses not lower than 8 cGy. Nevertheless, assuming a linear nonthreshold form for the overall dose-effect relationships of both radiation qualities, an RBE value of 12.3 is obtained for the 1.5 MeV neutrons. The induction of solid tumors by neutrons becomes statistically significant at individual doses from 8 cGy and by X rays for doses larger than 1 Gy. Linear dependence on neutron dose appears adequate to interpret the data at low doses. A separate analysis of ovarian tumor induction substantiates the hypothesis of a threshold dose for the X rays, while this is not strictly needed to interpret the neutron data. A trend analysis conducted on the neoplasm incidence confirms the above findings. Death rates have been analyzed, and a general agreement between the shift to earlier times of these curves and tumor induction was found.

Transplantation of harderian gland cells from CBA/-Cne mice into the fat pad of isogenic recipients was used for a quantitative in vivo study of cell survival and risk of transformation after x-ray irradiation (1-7 Gy). A survival curve for gland cells was generated in vivo with a D0 of 1.83 Gy and an extrapolation number of 7.23. Subsequently, the dose-response curve for lesions observed in nodules after cell transplantation was compared with that for lesions observed in glands irradiated in situ. A high incidence of epithelial hyperplasias with severe dysplasia was observed in transplantation nodules after x-irradiation. Gland tumors were significantly induced in whole-body irradiated animals; the tumors reached a maximum incidence after doses of 3 Gy. The risk of transformation per surviving cell was estimated both for dysplastic lesions and for tumors. These results approximated a dose-squared relationship in both cases, suggesting a common induction mechanism at the cellular level. Myeloid leukemia was observed at all doses in whole-body irradiated mice, and the maximum tumor incidence was reached at doses around 3 Gy.

Transplantation of hepatocytes from CBA/Cne mice into the fat pads of isogeneic recipients has been used for the quantitative in vivo study of cell survival and risk of transformation after x-ray irradiation (1-7 Gy). A survival curve for liver cells was generated in vivo with a D0 of 3.08 Gy and an extrapolation number not significantly different from 1. Data on liver tumor incidence in whole-body irradiated CBA/Cne and C57BL/Cne X C3H/HeCne (BC3F1) mice are also reported. A statistical analysis of trend in both cases proved a significant induction of tumors by x-rays mainly for doses above 2 Gy. The risk of transformation per surviving cell was estimated for both mouse strains. For CBA mice the data points suggested the presence of a linear component in the dose-effect curve at low doses, whereas for BC3F1 mice a quadratic expression appeared to provide a better description of the points from 1 to 6 Gy. The data of this study suggested that liver tumors can be induced by radiation in mouse strains with either a high or low spontaneous hepatoma incidence.

The main object of this study is to investigate the role of age on the susceptibility to radiation carcinogenesis and life shortening for different qualities of radiation. Over the last few years, a line of research at the Laboratory of Pathology, C.R.E. Casaccia, has been set up to study the effects of exposure to neutron irradiation, including observations on late effects (both neoplastic and nonneoplastic) as a function of radiation dose and of age at irradiation. Graded single doses of X rays or attenuated fission neutrons have been given to male BC3F1 mice 3 and 19 months old and to animals in utero at 17 days postcoitum. The analysis of data from over 3000 mice indicates that irradiation at 3 months of age causes life shortening which is associated with the incidence and rate of radiation-induced neoplasms. Prenatal irradiation or irradiation at 19 months of age does not show a clearly measurable life shortening for both X-ray and neutron exposures. However, significantly higher incidence and rate of solid tumors and reticulum cell sarcomas were observed. In general the data confirm the higher biological effectiveness of neutrons compared with X rays. The estimates of neutron relative biological effectiveness for different end points were found to be in the range of 3 to 18 and their variation was closely dose dependent.