p53 alterations in chemically induced hamster cheek‐pouch lesions

Abstract

To confirm that the hamster cheek-pouch carcinogenesis model reflects development of human squamous cell carcinoma (SCC), we determined if and when p53 mutations occur in the development of SCC in this model by using immunohistochemical staining and polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis plus direct DNA sequencing. Twenty-four hamster cheek-pouches were treated with a solution of 0.5% 7,12-dimethylbenz[a]anthracene in mineral oil three times a week for 16 wk. The malignant endophytic and exophytic tumors induced with this protocol are preceded by a sequence of premalignant lesions such as hyperplasia with or without dysplasia and carcinoma in situ, similar to the development of this cancer in humans. For this study, p53 protein accumulation was evaluated by immunostaining of various hamster cheek-pouch exophytic and endophytic SCCs as well as flat dysplastic hyperplasia and carcinomas in situ. A moderate percentage (33.3%) of exophytic lesions and most endophytic carcinomas (90%) showed positive p53 staining. In addition we also found p53-positive staining in a number of preneoplastic lesions, including areas of focal hyperplasia, dysplastic hyperplasia, and carcinomas in situ. To determine whether the alterations in p53 staining were due to p53 gene mutation, we used PCR-SSCP analysis and direct sequencing. PCR products corresponding to exons 5a, 6, 7, and 8 from 40 tumors with the highest percentage of p53-stained cells were analyzed. We detected shifted bands in 17 lesions. Direct sequencing of eight selected shifted bands revealed four mutations, including two G-->T transversions in codons 216 (tumor #1) and 252 (tumor #2) and one G-->C transversion in codon 282 (tumor #3). Tumor #4 contained a frameshift mutation in codon 251. These mutations are consistent with those reported in many human cancers. Therefore, we concluded that in the hamster cheek-pouch model, p53 protein accumulation occurs frequently and early in carcinogenesis, as it does in human SCCs, and some of these p53 alterations are due to p53 gene mutations. These findings may help us better define the mechanisms of carcinogenesis in the hamster cheek-pouch model, and p53 alterations may be an early biomarker of progression for chemoprevention studies.

Cytologic alterations in the oral mucosa after chronic exposure to ethanol

“…The buccal mucosa showed far fewer significant results, although it is also lined by stratified nonkeratinized squamous epithelium. These findings agree with previous literature reports that point out the buccal mucosa as being a region less exposed to carcinogenic action 8 . The presence of contaminants, such as polycyclic aromatic hydrocarbons and nitrosamines, is a significant carcinogenic factor in alcoholic beverages 26 .…”

Cytologic alterations in the oral mucosa after chronic exposure to ethanol

“…The buccal mucosa showed far fewer significant results, although it is also lined by stratified nonkeratinized squamous epithelium. These findings agree with previous literature reports that point out the buccal mucosa as being a region less exposed to carcinogenic action 8 . The presence of contaminants, such as polycyclic aromatic hydrocarbons and nitrosamines, is a significant carcinogenic factor in alcoholic beverages 26 .…”

Correlation between inducible nitric oxide synthase and p53 expression for DMBA‐induced hamster buccal‐pouch carcinomas

“…Therefore, the present study further supports the hypothesis that iNOS may be associated with oral carcinogenesis. The result of this study is in agreement with two earlier reports regarding p53 expression for chemically induced hamster-pouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000). Here, using RT-PCR, we report that p53 mRNA has been over-expressed amongst DMBA-induced hamster buccal-pouch carcinomas.…”

“…Further, from the work by Forrester et al (1996), the cellular accumulation of wild-type p53 was found to suppress iNOS genepromoter activity, resulting in the down-regulation of iNOS expression, thus suggesting a negative feedback loop between iNOS expression and wild-type p53. Thus, based on these findings, along with the appreciation of a rather high p53 mutation frequency for hamster buccalpouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000) and the aforementioned results of this study, two possible mechanisms may be associated with the correlation between iNOS and p53 expression for DMBA-induced hamster buccal-pouch carcinogenesis.…”

Differential expression of p53, p63 and p73 protein and mRNA for DMBA‐induced hamster buccal‐pouch squamous‐cell carcinomas

“…Although the expression of p73, p63 and p53 in hamster buccal-pouch carcinomas has been investigated previously by our group (Chen et al 2002b(Chen et al , 2003a(Chen et al , 2003b as also other workers (Chang et al 1996;Gimenez-Conti et al 1996), it would appear, to the best of our knowledge, that the differential expression for all of the p53 homologues, namely, p73, p63 and p53 for chemically induced experimental oral carcinomas has not been previously reported. In this study, the differential expression of p73, p63 and p53 protein and mRNA was characterized for a subset of DMBA-induced squamous-cell carcinomas arising from hamster buccalpouch buccal mucosa, with further elucidation of the expression for all of the p53 homologues in an experimental oral carcinoma model.…”

Cytologic alterations in the oral mucosa after chronic exposure to ethanol

“…The buccal mucosa showed far fewer significant results, although it is also lined by stratified nonkeratinized squamous epithelium. These findings agree with previous literature reports that point out the buccal mucosa as being a region less exposed to carcinogenic action 8 . The presence of contaminants, such as polycyclic aromatic hydrocarbons and nitrosamines, is a significant carcinogenic factor in alcoholic beverages 26 .…”

Correlation between inducible nitric oxide synthase and p53 expression for DMBA‐induced hamster buccal‐pouch carcinomas

“…Therefore, the present study further supports the hypothesis that iNOS may be associated with oral carcinogenesis. The result of this study is in agreement with two earlier reports regarding p53 expression for chemically induced hamster-pouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000). Here, using RT-PCR, we report that p53 mRNA has been over-expressed amongst DMBA-induced hamster buccal-pouch carcinomas.…”

“…Further, from the work by Forrester et al (1996), the cellular accumulation of wild-type p53 was found to suppress iNOS genepromoter activity, resulting in the down-regulation of iNOS expression, thus suggesting a negative feedback loop between iNOS expression and wild-type p53. Thus, based on these findings, along with the appreciation of a rather high p53 mutation frequency for hamster buccalpouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000) and the aforementioned results of this study, two possible mechanisms may be associated with the correlation between iNOS and p53 expression for DMBA-induced hamster buccal-pouch carcinogenesis.…”

Differential expression of p53, p63 and p73 protein and mRNA for DMBA‐induced hamster buccal‐pouch squamous‐cell carcinomas

“…Although the expression of p73, p63 and p53 in hamster buccal-pouch carcinomas has been investigated previously by our group (Chen et al 2002b(Chen et al , 2003a(Chen et al , 2003b as also other workers (Chang et al 1996;Gimenez-Conti et al 1996), it would appear, to the best of our knowledge, that the differential expression for all of the p53 homologues, namely, p73, p63 and p53 for chemically induced experimental oral carcinomas has not been previously reported. In this study, the differential expression of p73, p63 and p53 protein and mRNA was characterized for a subset of DMBA-induced squamous-cell carcinomas arising from hamster buccalpouch buccal mucosa, with further elucidation of the expression for all of the p53 homologues in an experimental oral carcinoma model.…”

Cytologic alterations in the oral mucosa after chronic exposure to ethanol

“…The buccal mucosa showed far fewer significant results, although it is also lined by stratified nonkeratinized squamous epithelium. These findings agree with previous literature reports that point out the buccal mucosa as being a region less exposed to carcinogenic action 8 . The presence of contaminants, such as polycyclic aromatic hydrocarbons and nitrosamines, is a significant carcinogenic factor in alcoholic beverages 26 .…”

Correlation between inducible nitric oxide synthase and p53 expression for DMBA‐induced hamster buccal‐pouch carcinomas

“…Therefore, the present study further supports the hypothesis that iNOS may be associated with oral carcinogenesis. The result of this study is in agreement with two earlier reports regarding p53 expression for chemically induced hamster-pouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000). Here, using RT-PCR, we report that p53 mRNA has been over-expressed amongst DMBA-induced hamster buccal-pouch carcinomas.…”

“…Further, from the work by Forrester et al (1996), the cellular accumulation of wild-type p53 was found to suppress iNOS genepromoter activity, resulting in the down-regulation of iNOS expression, thus suggesting a negative feedback loop between iNOS expression and wild-type p53. Thus, based on these findings, along with the appreciation of a rather high p53 mutation frequency for hamster buccalpouch carcinomas (Gimenez-Conti et al, 1996;Chang et al, 2000) and the aforementioned results of this study, two possible mechanisms may be associated with the correlation between iNOS and p53 expression for DMBA-induced hamster buccal-pouch carcinogenesis.…”

Differential expression of p53, p63 and p73 protein and mRNA for DMBA‐induced hamster buccal‐pouch squamous‐cell carcinomas

“…Although the expression of p73, p63 and p53 in hamster buccal-pouch carcinomas has been investigated previously by our group (Chen et al 2002b(Chen et al , 2003a(Chen et al , 2003b as also other workers (Chang et al 1996;Gimenez-Conti et al 1996), it would appear, to the best of our knowledge, that the differential expression for all of the p53 homologues, namely, p73, p63 and p53 for chemically induced experimental oral carcinomas has not been previously reported. In this study, the differential expression of p73, p63 and p53 protein and mRNA was characterized for a subset of DMBA-induced squamous-cell carcinomas arising from hamster buccalpouch buccal mucosa, with further elucidation of the expression for all of the p53 homologues in an experimental oral carcinoma model.…”