Biology Contribution
Genetic Background Modulates Gene Expression Profile Induced by Skin Irradiation in Ptch1 Mice

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Purpose

Ptch1 germ-line mutations in mice predispose to radiation-induced basal cell carcinoma of the skin, with tumor incidence modulated by the genetic background. Here, we examined the possible mechanisms underlying skin response to radiation in F1 progeny of Ptch1neo67/+ mice crossed with either skin tumor-susceptible (Car-S) or -resistant (Car-R) mice and X-irradiated (3 Gy) at 2 days of age or left untreated.

Methods and Materials

We conducted a gene expression profile analysis in mRNA samples extracted from the skin of irradiated or control mice, using Affymetrix whole mouse genome expression array. Confirmation of the results was done using real-time reverse-transcriptase polymerase chain reaction.

Results

Analysis of the gene expression profile of normal skin of F1 mice at 4 weeks of age revealed a similar basal profile in the nonirradiated mice, but alterations in levels of 71 transcripts in irradiated Ptch1neo67/+ mice of the Car-R cross and modulation of only eight genes in irradiated Ptch1neo67/+ mice of the Car-S cross.

Conclusions

These results indicate that neonatal irradiation causes a persistent change in the gene expression profile of the skin. The tendency of mice genetically resistant to skin tumorigenesis to show a more complex pattern of transcriptional response to radiation than do genetically susceptible mice suggests a role for this response in genetic resistance to basal cell tumorigenesis.

Introduction

Inherited mutations of the Patched homolog 1 (Drosophila) tumor suppressor gene in human beings predispose to nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominantly inherited disorder associated with the development of multiple basal cell carcinomas (BCCs) (1), occurring with greater incidence in portals of radiotherapy.

The risk of the sporadic form of this tumor type is modulated by genetic predisposition, as individuals with first-degree relatives affected by BCCs have an increased risk of developing this tumor with respect to the general population (2), although the responsible factors remain unknown. Ionizing radiation is also an important and established risk factor for BCCs in both sporadic cases and in NBCCS patients (3).

Patched (Ptch1) heterozygous mice recapitulate the etiology and the histopathology of human BCC, and offer opportunities to investigate the role of genetic background in radiation-induced BCC susceptibility. Indeed, crosses between the Patched (Ptch1) gene knock-out mouse and outbred mouse lines that are skin carcinogenesis resistant and susceptible (Car-R and Car-S, respectively) (4) show different responses to radiation-induced BCC (5). Ptch1 knock-out mice are also particularly susceptible to radiation-induced medulloblastomas (6). To study the early changes associated with radiation-induced BCC in different genetic backgrounds, we examined the gene expression profile of the normal or irradiated skin of F1S Ptch1neo67/+ and F1R Ptch1neo67/+ mice generated by crossing Ptch1 heterozygous gene knock-out (Ptch1neo67/+) males on a CD1 background with Car-S and Car-R female mice (6). Our results identify several candidate genes and pathways that might be involved in genetic resistance or susceptibility to radiation-induced skin tumorigenesis. Our results point to the potential role of transcriptional changes in conferring resistance to radiation-induced skin tumorigenesis.

Section snippets

Mice and treatment

Mice lacking one Ptch1 allele (Ptch1neo67/+), derived by gene targeting of 129Sv embryonic stem cells, were maintained on the outbred CD1 strain background (5) and crossed with either Car-R or Car-S mice to obtain F1 progeny lacking one Ptch1 allele (designated F1R and F1S, respectively) (Fig. 1). Groups of 8-10 F1R and F1S mice were whole-body irradiated with a single dose of 3 Gy X-radiation at 2 days of age as described (5). Control groups were left untreated. Mice were sacrificed at 4 weeks

Results

RNAs derived from all the samples were of good quality, since their RNA Integrity Number (RIN) ranged from 7.3 to 9.4. Basal transcript levels in nonirradiated F1R and F1S mice were similar, with only six known genes (H2-D1, LOC56628, Mela, Oasl2, Scd2, Slc13a3) and 3 unknown transcripts (2810417H13Rik, 9230117E20Rik, C430014N20Rik) showing fourfold or greater differences.

In the skin of irradiated F1R mice, 71 transcripts were differentially expressed (fourfold or greater change), 66 of which

Discussion

Fig. 2, Fig. 3 show examples of 10 genes the mRNA levels of which modulated by radiation in a genetic background-dependent way. Indeed, in both F1R and F1S mice, these genes were up- or downregulated in adult skin after neonatal whole-body radiation treatment, although the modulation was more pronounced in F1R (Fig. 2) than in F1S mice (Fig. 3). Some of these genes participated in multiple pathways pointed out by the gene expression profile analysis (Table 3). Desmocollin 2 (Dsc2) gene is

Conclusion

In the present study, we find that radiation induces a persistent change in the gene expression profile in mice genetically resistant to skin carcinogenesis but only a minimal response in the genetically susceptible mice. These results raise the possibility that a transcriptional response to radiation represents a mechanism by which the organism counteracts radiation effects that could lead to BCC tumorigenesis. Thus, genetic susceptibility to radiation-induced BCC may rest in the lack of an

Acknowledgments

We thank the BioGeM Gene Expression Core (Ariano Irpino) for the expression profile analysis. This work was funded in part by grants from Associazione and Fondazione Italiana Ricerca Cancro (AIRC and FIRC) and by a grant (RISC-RAD) from the European Commission.

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