Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Winter temperature and UV are tightly linked to genetic changes in the p53 tumor suppressor pathway in Eastern Asia.

The tumor suppressor p53 is a master sensor of stress. Two human-specific polymorphisms, p53 codon 72 and MDM2 SNP309, influence the activities of p53. There is a tight association between cold winter temperature and p53 Arg72 and between low UV intensity and MDM2 SNP309 G/G in a cohort of 4029 individuals across Eastern Asia that suggests causative selection. Moreover, the two polymorphisms are not coselected. Haplotype-based selection analysis further suggests that this is a striking example of two functional polymorphisms being strongly selected for in human populations in response to environmental stresses.

Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Possible role of a radiation-induced p53 mutation in a Nelson's syndrome patient with a fatal outcome.

Nelson's syndrome (NS) is characterized by the appearance and/or progression of ACTH-secreting pituitary macroadenomas in patients who had previously undergone bilateral adrenalectomy for the treatment of Cushing's disease. Such corticotroph macroadenomas respond poorly to currently available therapeutic options which include surgery, radiotherapy and chemotherapy. P53 protein accumulation may be detected by immunohistochemistry in pituitary corticotroph adenomas and it has been suggested that it might be causally related to tumor development. Wild type P53 protein plays an important role in the cellular response to ionizing radiation and other DNA damaging agents and is mutated in many human tumors. In this study we report an adult male patient with NS who underwent both transsphenoidal and transcranial pituitary surgeries, conventional and stereotaxic radiotherapy and brachytherapy. Despite of the efforts to control tumor mass and growth, this macroadenoma displayed relentless growth and aggressive behavior. DNA extracted from the first two surgical samples, as well as DNA from peripheral blood leukocytes disclosed normal p53 sequence. DNA extracted from tumor samples obtained at surgeries performed after pituitary irradiation carried a somatic heterozygous mutation, consisting of a deletion of four cytosines between nucleotides 12,144-12,149 in exon 4 of the p53 gene. This frameshift mutation creates a stop codon in exon 4 excluding the expression of a functional protein from the defective allele. These data demonstrate a possible association between the P53 protein loss of function induced by radiotherapy and the aggressive course of the disease in this patient.

[The comparative determination mutations in mtDNA of irradiated mice using mismatch-specific endonuclease and TTGE-technique].

We defined the mutations in mtDNA of X-irradiated mice brair using mismatch-specific endonuclease (CEL I-nuclease method) and by temporal temperature gradient gel electrophoresis (TTGE-technique). The comparison of the received by both methods, allows to conclude, that CEL I-nuclease method gives more qualitative results, than TTGE-technique. Moreover, CEL I-nuclease method is more sensitive, in contrast with TTGE-technique. The CEL I-nuclease method allows simultaneously to conduct the analysis of big amount of sample DNA, to get the reproducible results. It does not require complex equipment and economical. The analysis of mutations in mtDNA of brain of X-irradiated mice by CEL I-nuclease method has shown, that the amount of mutant copies mtDNA is essentially reduced (in 2-3 times) with 8 up to 28 days of the post-radiation period. However the amount mtDNA copies in brain tissue of the irradiated animals is remains during all post radiation time without change though lower, concerning given control group. The results permit the suggestion that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the post-radiation period.

Gene expression profiling of breast, prostate, and glioma cells following single versus fractionated doses of radiation.

Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 x 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-beta-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures.

RB1 and TP53 pathways in radiation-induced sarcomas.

The tumour suppressor genes, TP53 and RB1, and four genes involved in their regulation, INK4a, ARF, MDM2 and MDMX, were analysed in a series of 36 post-radiotherapy radiation-induced sarcomas. One-third of the tumours developed in patients carrying a germline mutation of RB1 that predisposed them to retinoblastoma and radiation-induced sarcomas. The genetic inactivation of RB1 and/or TP53 genes was frequently observed in these sarcomas. These inactivations were owing to an interplay between point mutations and losses of large chromosome segments. Radiation-induced somatic mutations were observed in TP53, but not in RB1 or in the four other genes, indicating an early role of TP53 in the radio-sarcomagenesis. RB1 and TP53 genes were biallelically coinactivated in all sarcomas developing in the context of the predisposition, indicating that both genes played a major role in the formation of these sarcomas. In the absence of predisposition, TP53 was biallelically inactivated in one-third of the sarcomas, whereas at least one allele of RB1 was wild type. In both genetic contexts, the TP53 pathway was inactivated by genetic lesions and not by the activation of the ARF/MDM2/MDMX pathway, as recently shown in retinoblastomas. Together, these findings highlight the intricate tissue- and aetiology-specific relationships between TP53 and RB1 pathways in tumorigenesis.

Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis.

PURPOSE: The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis). MATERIALS AND METHODS: Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5 degrees C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion. RESULTS: Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines. CONCLUSION: These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.

Enhanced photocarcinogenesis in interleukin-12-deficient mice.

UV-induced DNA damage is the basis for the development of UV-mediated skin cancer because reduction of DNA damage lowers the risk for photocarcinogenesis. The cytokine interleukin (IL)-12 was shown to exhibit the capacity to reduce UV-induced DNA damage presumably via induction of nucleotide excision repair. Because IL-12 is also produced in the skin, we wondered whether endogenous IL-12 protects from photocarcinogenesis. Therefore, we used knockout mice that lack the IL-12p40 chain and thus do not secrete biologically active IL-12. IL-12p40 knockout (IL-12p40-/-) and wild-type (wt) mice were exposed thrice weekly to UV. Skin biopsies obtained after 6 weeks revealed significantly increased numbers of sunburn cells in IL-12p40-/- mice. Additionally, a higher load of UV-induced pyrimidine dimers could be detected in the skin of UV-exposed IL-12p40-/- mice. Staining of epidermal sheets with an antibody against the tumor suppressor gene p53 revealed a higher number of p53 patches in the skin of IL-12p40-/- mice. After approximately 200 days, first skin tumors developed. Kaplan-Meier analysis indicated a significantly increased probability of tumor development in the IL-12p40-/- mice. In addition, the number of tumors developing in the individual mice was significantly higher in IL-12p40-/- mice than in wt mice. Tumors obtained in IL-12p40-/- mice grew faster than those obtained from wt mice on inoculation into nu/nu mice. This was confirmed in an electrophysiologic assay evaluating the intrinsic invasive potency of tumor cells. Together, these data indicate that IL-12 deficiency is associated with an increased risk to develop UV-induced skin cancer, implying that endogenous IL-12 may protect from photocarcinogenesis.

Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.

The changing face of p53 in head and neck cancer.

p53 plays a sentinel role in the pathways that prevent development of cancer by inducing apoptosis, DNA repair and cell-cycle arrest in response to different types of cellular stress. The majority of head and neck tumours harbour mutations affecting the p53 gene, and those tumours that seemingly have wild-type p53 protein most probably lack a functional p53 response as a result of mutations affecting other genes that function in the same pathways as p53. This report provides an up-to-date overview of what is known about how p53 exerts its effects. We also summarize what is known about the other p53 family members, p63 and p73, and show how they act together to influence the response to treatment. No other commonly occurring signature mutation has emerged for this tumour type, and this means that the p53 family has emerged as the frontrunner in terms of providing molecular targets that can provide new diagnostic, prognostic and therapeutic approaches.

Patches of mutant p53-immunoreactive epidermal cells induced by chronic UVB Irradiation harbor the same p53 mutations as squamous cell carcinomas in the skin of hairless SKH-1 mice.

Treatment of SKH-1 hairless mice with UVB (30 mJ/cm(2)) twice a week for 20 weeks results in the formation of cellular patches, long before the appearance of tumors, that are visualized in epidermal sheets with an antibody (PAb240) recognizing mutated p53 protein. Direct sequencing analysis of the whole coding region of the p53 gene (exons 2-11) detected one or two mutations in 64.4% of 104 analyzed patches and no mutations in nonstained adjacent normal controls. Homozygous mutation was detected in 22.4% of the mutant patches. Except for two nonsense mutations, all others were missense (exons 4-9) and mostly (95.5%) at the DNA-binding domain. Primer extension analysis of cloned PCR fragments found three of four double-mutated patches harboring different mutations in separate alleles. All mutation hotspots reported earlier in UVB-induced mouse squamous cell carcinomas (SCC) at codons 270 (Arg --> Cys), 149 (Pro --> Ser), 275 (Pro --> Leu and Pro --> Ser), and 176 (His --> Tyr) with a frequency of 32.1%, 7.1%, 14.7%, and 3.2% were detected in epidermal patches at a frequency 47.7%, 9.1%, 4.5%, and 2.3%, respectively. Mutations at codons 210 and 191 found in patches at respective frequencies of 8.0% and 4.5% were not previously detected in UVB-induced mouse SCC. In summary, (a) the p53 mutation profile of UVB-induced skin patches and SCC was very similar suggesting that patches are precursor lesions for SCC, (b) a small number of patches harbored mutations that were not before observed in SCC from UVB-treated mice, and (c) about 36% of the patches did not harbor a p53 mutation.

Lack of p53 Ser389 phosphorylation predisposes mice to develop 2-acetylaminofluorene-induced bladder tumors but not ionizing radiation-induced lymphomas.

Cellular activity of the tumor suppressor protein p53 is primarily regulated by posttranslational modifications. Phosphorylation of the COOH terminus, including Ser389, is thought to result in a conformational change of the p53 protein, enhancing DNA binding and transcriptional activity. In vitro studies presented here show that, in addition to UV radiation, Ser389 is phosphorylated upon exposure to 2-acetylaminofluorene (2-AAF). Both agents induce bulky DNA adducts repaired by nucleotide excision repair (NER). In contrast, ionizing radiation, known to induce DNA damage not repaired by NER, does not result in Ser389 phosphorylation. Previously, we have shown that p53.S389A mutant mice, lacking the Ser389 phosphorylation site, are sensitive to developing UV-induced skin tumors. Here, we show that p53.S389A mice are also prone to developing 2-AAF-induced urinary bladder tumors, whereas no increased tumor response was found upon ionizing irradiation. These results provide evidence for our hypothesis that phosphorylation of Ser389 is important for activation of p53 to exert its function as a tumor suppressor not exclusively upon the presence of UV-induced DNA damage, but also upon exposure to other bulky adduct-inducing agents. Analysis of 2-AAF- and UV-induced tumors from p53.S389A mice revealed the presence of additional p53 mutations, indicating that lack of Ser389 phosphorylation by itself is not sufficient to abrogate p53 function in tumor suppression. In addition, analyses of skin tumors of p53.S389A mice revealed an interesting hotspot mutation previously found exclusively in NER-deficient mice and patients.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer.

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line.

Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mug/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.

Lymphoblastoid cell lines differing in p53 status show clear differences in basal gene expression with minor changes after irradiation.

BACKGROUND AND PURPOSE: The genetic profile as determined by microarray is considered to be an ideal marker of the individual radiosensitivity. However, it is still an open question, whether this profile has to be determined prior to or only after irradiation, since the expression of some genes is affected by irradiation. These changes are induced mainly due to a p53-dependent transactivation. MATERIALS AND METHODS: In this study gene expression profiles were measured for 3 lymphoblastoid cell lines differing in p53 status (p53 wt: TK6; p53null: TK6E6, p53mut: WTK1) measured either prior to or 3h after exposure to 2Gy. The gene expression profile was determined using the Affymetrix Human HG U133A GeneChip and for selective genes, variation in gene expression was validated by qRT-PCR. In addition, different assays were used to characterize the radioresponse of these three strains. RESULTS: The three strains were found to be different in all aspects of radiosensitivity studied. Cells with p53wt showed more apoptosis, slightly stronger arrest in G1, but less lethal aberrations and a lower viability when compared to cells with mutated p53, whereas cells absent in p53 are characterized by an intermediate response. The gene expression profile measured prior to irradiation already revealed huge differences. Significance analysis of microarrays (SAM) identified 141 genes that changed expression twofold or more with a false discovery rate (FDR) of 5.4%. When compared to p53null cell line with p53wt showed a twofold difference in up- or down-regulation in 28 genes. A much higher variation was even found when p53mut cells were compared with p53null cells with a twofold difference in even 123 genes. The respective genes were found to be involved mainly in apoptosis, cell cycle regulation, metabolisms and signalling but with only one gene relevant for DNA repair. Radiation was found to affect this profile solely for cells with p53wt with a twofold significant up-regulation in only five genes. For selective genes (BCL2, CASP1, CCND2, DDB2, XPC, RAD51C, SESN1, FUCA1, CDKN1A, MDM2, XPC) array data were confirmed by qRT-PCR. CONCLUSION: The result, that the gene expression profile of lymphoblastoid cells differing in p53 status already displayed clear differences when measured prior to irradiation with only few changes after irradiation, which are solely seen for p53wt cells, suggests, that the differences in radiosensitivity observed for these cells are primarily determined by the variation in expression profile present already prior to irradiation.

Radiation-induced effects on gene expression: an in vivo study on breast cancer.

BACKGROUND AND PURPOSE: Breast cancer is diagnosed worldwide in approximately one million women annually and radiation therapy is an integral part of treatment. The purpose of this study was to investigate the molecular basis underlying response to radiotherapy in breast cancer tissue. MATERIAL AND METHODS: Tumour biopsies were sampled before radiation and after 10 treatments (of 2 Gray (Gy) each) from 19 patients with breast cancer receiving radiation therapy. Gene expression microarray analyses were performed to identify in vivo radiation-responsive genes in tumours from patients diagnosed with breast cancer. The mutation status of the TP53 gene was determined by using direct sequencing. RESULTS AND CONCLUSION: Several genes involved in cell cycle regulation and DNA repair were found to be significantly induced by radiation treatment. Mutations were found in the TP53 gene in 39% of the tumours and the gene expression profiles observed seemed to be influenced by the TP53 mutation status.

Analysis of p53 mutations for a mutational signature in human intrahepatic cholangiocarcinoma.

Cholangiocarcinoma development may be related to cholangiocyte DNA damage from genotoxic compounds in bile. We have previously shown that human biliary tissue is exposed to genotoxic agents, as evidenced by the presence of DNA adducts. Establishing the presence of a 'mutational signature' in tumour suppressor genes from tumour tissue provides a means of linking cause and effect in human cancer. Inactivation of p53, known to have 'hot-spots' for particular chemical carcinogens, has previously been linked to human cholangiocarcinoma. However, previous p53 studies have focused on exons 5-8, potentially missing gene alterations at other sites. This study examined the putative link between environmental carcinogens and intrahepatic cholangiocarcinoma by analysing DNA from 31 patients for complete p53 mutational signatures, using single strand conformational polymorphism and polymerase chain reaction. All mutations found were compared to known p53 mutations in cholangiocarcinoma and to mutations induced by environmental mutagens, as described in p53 databases. Five non-silent p53 mutations were found, including three new frameshift mutations and two new intron mutations which have not previously been reported in cholangiocarcinoma. Two frameshifts were due to deletions and the third due to an insertion in exon 5. There was no predominant mutational spectrum amongst the set of cholangiocarcinoma samples studied, or on combining these mutations with the dataset of known p53 mutations in cholangiocarcinoma. Several reasons may explain this, including lack of data outside exons 5-8, bias in mutation reporting, the involvement of mutations in non-coding regions or genes other than p53, or the possibility that there is no carcinogenic specific agent and therefore no signature.

Ultraviolet radiation induction of squamous cell carcinomas in p53 transgenic mice.

Mutations of the p53 gene have been implicated in the pathogenesis of cutaneous squamous cell carcinoma (SCC). To examine the role of p53 in skin carcinogenesis, we observed the development of skin cancers in p53 transgenic mice which carry multiple copies of a mutant allele of the p53 gene with or without chronic UVB radiation. Thirty-one tumors developed in 19 UV-irradiated p53 transgenic mice versus 14 of 19 in the control group; 9 p53 transgenic mice but none of the control mice developed multiple tumors. Histologically. 14 of 14 tumors in the CD-1 mice were SCCs. In the p53 transgenic mice, 25 of 31 tumors were SCCs, and 6 were benign tumors. The mean time to appearance of tumors did not differ between CD-1 mice (26.3 weeks) and the p53 transgenic mice (25.7 weeks; P = 0.512). The p53 protein, which was undetectable by immunohistochemistry in the keratinocytes of CD-1 mice, was elevated in 93% (13 of 14) of tumors from CD-1 mice. These data indicate that mutation of the p53 gene is an important step in the development of SCC. p53 mutations do not alter the latent period of UV-induced SCC but significantly increase the number of tumors and the propensity for multiple tumor development.

Latent expression of p53 mutations and radiation-induced mammary cancer.

EF42 is a clonally derived preneoplastic cell lineage from irradiated mouse mammary tissue, which becomes neoplastic with time in vitro or in vivo. We now report that multiple mutations in p53 occur before the acquisition of the neoplastic phenotype. The selective expansion of mutant cells is accompanied by loss of heterozygosity at the p53 locus and c-myc amplification. Although p53 mutations represent critical early events, our data argue these mutations were not directly induced by radiation but arose in the progeny of irradiated cells several cell generations later. The data are consistent with a multistep model of carcinogenesis that identifies genomic instability as the earliest step.