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Deletion of P15 (MTS2) in head and neck squamous cell carcinomas
J M Roesler1, E H Livingston, E Srivatsan
1Department of General Surgery, UCLA School of Medicine, Los Angeles, California, 90095-6904, USA.
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.
Insights
Nearly 50% of head and neck squamous cell carcinomas (HNSCC) showed absence of the p15 (MTS2) gene. Loss of this gene may impact cell cycle regulation in HNSCC.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Head and neck squamous cell carcinomas (HNSCC) frequently exhibit loss of heterozygosity (LOH) on chromosome 9p21-p22, a region implicated in tumor suppression.
- The p16 (MTS1) gene, an inhibitor of cyclin-dependent kinases 4 and 6 (cdk4/cdk6), is located in this region and frequently deleted.
- The p15 (MTS2) gene, also an inhibitor of cdk4/cdk6 with similar biochemical properties to p16, has not been fully characterized in HNSCC.
Purpose of the Study:
- To investigate the deletion frequency of the multiple tumor suppressor 2 (MTS2) gene, which encodes the p15 protein, in head and neck squamous cell carcinomas (HNSCC).
- To determine if the p15 (MTS2) gene is altered in HNSCC, similar to the frequently deleted p16 (MTS1) gene in the same chromosomal region.
Main Methods:
- Analysis of 21 head and neck squamous cell carcinomas (HNSCC) and their proximal margins using snap freezing and standard DNA isolation techniques.
- Polymerase Chain Reaction (PCR) with primers specific for exon 1 of the p15 gene was employed.
- Amplification of the beta-Globin gene served as a positive control to ensure DNA quality, with PCR products analyzed on polyacrylamide gels.
Main Results:
- All 21 proximal margins showed normal amplification of the p15 gene, with a visible 532 bp PCR product.
- Nine out of 21 (approximately 43%) HNSCC tumors exhibited no amplification of the p15 gene, lacking visible PCR products.
- All samples, including tumors and margins, demonstrated beta-Globin amplification, confirming the integrity of the DNA used in the study.
Conclusions:
- The study demonstrates the absence of normal p15 (MTS2) exon 1 gene amplification in nearly 50% of the studied HNSCC cases.
- Densitometric analysis suggests that even in tumors with detectable PCR product, the p15 gene is present in a small fraction of tumor cells (less than 3% compared to normal cells).
- Loss or significant reduction of the p15 (MTS2) gene product may play a role in the dysregulation of cell cycle and growth observed in head and neck squamous cell carcinomas.