The Journal of surgical research · 1998
Deletion of P15 (MTS2) in head and neck squamous cell carcinomas.
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Roesler J M, Livingston E H, Srivatsan E, Chang P, Wang M B
Аннотация
INTRODUCTION: The purpose of this study was to determine whether the multiple tumor suppressor 2 (MTS2) gene, encoding an inhibitor (p15) of cyclin D-dependent kinases 4 and 6 (cdk4, cdk6), is deleted in head and neck squamous cell carcinomas (HNSCC). There is a high frequency of LOH for the 9p21-p22 region in HNSCCs, as well as in gliomas, leukemias, and cell lines from multiple tumor types; thus, this region is suspected to contain a tumor suppressor gene or genes. P16 (MTS1), an inhibitor of cdk4 and cdk6, resides within the deleted 9p21 region in these tumors. A neighboring gene, p15 (MTS2), has biochemical properties similar to those of p16, but has not been characterized in HNSCC.
METHODS: Twenty-one head and neck squamous cell carcinomas and their proximal margins were snap frozen at the time of surgical resection. DNA isolation was performed using standard phenol and chloroform extraction. Standard PCR methods were used with primers P15-1F and P15-1R, specific for exon 1 of the p15 gene, as described previously. All samples were amplified for beta-Globin as a positive control. PCR products were stained with ethidium bromide and run on 6% polyacrylamide gels. Expected sizes for the PCR products were p15, 532 bp, and beta-globin, 238 bp. Results. Of 21 proximal margins, all demonstrated normal amplification of p15 DNA, all having a visible 532bp PCR product. Of 21 HNSCC tumors, 9 showed no amplification of the p15 gene; none of these 9 neoplasms had visible PCR products. All proximal margins and head and neck squamous cell carcinomas demonstrated amplification of the beta-globin gene, indicating that the DNA used was of good quality.
CONCLUSIONS: Although PCR is not a quantitative technique, densitometric analysis of PCR products showed it was unlikely that the p15 gene was present in more than a small fraction of the tumor cells. The amount of the p15 PCR product in these cells was less than 3% of that observed in reactions containing an equal amount of DNA from normal cells. We are the first to show an absence of normal p15 exon 1 gene amplification in nearly 50% of HNSCCs studied. Loss of the MTS2 gene product, p15, may contribute to the loss of cell cycle and growth regulation seen in HNSCC.
METHODS: Twenty-one head and neck squamous cell carcinomas and their proximal margins were snap frozen at the time of surgical resection. DNA isolation was performed using standard phenol and chloroform extraction. Standard PCR methods were used with primers P15-1F and P15-1R, specific for exon 1 of the p15 gene, as described previously. All samples were amplified for beta-Globin as a positive control. PCR products were stained with ethidium bromide and run on 6% polyacrylamide gels. Expected sizes for the PCR products were p15, 532 bp, and beta-globin, 238 bp. Results. Of 21 proximal margins, all demonstrated normal amplification of p15 DNA, all having a visible 532bp PCR product. Of 21 HNSCC tumors, 9 showed no amplification of the p15 gene; none of these 9 neoplasms had visible PCR products. All proximal margins and head and neck squamous cell carcinomas demonstrated amplification of the beta-globin gene, indicating that the DNA used was of good quality.
CONCLUSIONS: Although PCR is not a quantitative technique, densitometric analysis of PCR products showed it was unlikely that the p15 gene was present in more than a small fraction of the tumor cells. The amount of the p15 PCR product in these cells was less than 3% of that observed in reactions containing an equal amount of DNA from normal cells. We are the first to show an absence of normal p15 exon 1 gene amplification in nearly 50% of HNSCCs studied. Loss of the MTS2 gene product, p15, may contribute to the loss of cell cycle and growth regulation seen in HNSCC.