Original articles
Abnormalities of Tumor Suppressor Genes P16 and P15 in Primary Maxillofacial Squamous Cell Carcinomas

https://doi.org/10.1016/S0165-4608(98)00259-3Get rights and content

Abstract

As members of the same gene family, tumor suppressor genes P16/CDKN2/INK4A and P15/INK4B have a high degree of structural and functional homology with both P16 and P15 proteins involved directly in the regulation of cell cycles. However, the status of P16 and P15 genes in primary maxillofacial squamous cell carcinomas (MSCC) has not been reported. Studies on abnormalities of these genes including homozygous deletion, methylation of the 5CpG islands, and mutations were carried out in 65 primary MSCC with polymerase chain reaction (PCR), methylation-specific PCR (MSP), PCR-SSCP (single-strand conformation polymorphism), and DNA sequencing techniques. Of the 65 tumors, 22 (34%) were methylated; 7 (11%) displayed point mutations. The total frequency of alteration of the P16 gene was 43% (28/65). The methylation rate of P15 was 12% (8/65). No homozygous deletion was found in either the P16 gene or P15 gene. In all MSCC samples, almost half (49%) harbored an alteration of the P16 or P15 gene. The P16 gene was altered more frequently than P15, and therefore is inactivated by methylation or mutation in a significant proportion of MSCC. The P15 gene appeared to play a lesser role in tumorigenesis of these tumors.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is one of the most common human cancers. Several types of genetic alterations have been reported to contribute to the tumorigenesis of HNSCC, including mutational inactivation of the tumor suppressor gene TP53 and amplification of epidermal growth factor receptor, cyclin D 1, 2. Loss of heterozygosity (LOH) in several chromosomal regions was also described [3], and it is remarkable that about 70% of these neoplasms show LOH at 9p21, where the tumor suppressor genes P16/CDKN2/INK4A and P15/INK4B are located [4].

Both P16 and P15 genes belong to the inhibitors of the cyclin-dependent kinase 4 (INK4) family. The P16 gene encodes a cell cycle protein which inhibits cyclin dependent kinases 4 and 6 (CDK4 and 6), preventing phosphorylation of Rb protein and inhibiting cell cycle progression from G1 to the S phase [5]. Genetic alterations of the P16 gene, leading to its inactivation, may result in deregulation of cell proliferation and tumorigenesis. The gene is frequently altered in most human cancers, including HNSCC. In the latter, however, the incidence of P16 gene mutations and homozygous deletions in primary tumors is less than that in cell lines [6]. Recently, another mechanism of P16 inactivation, the methylation of the 5′CpG island of the gene, has also been reported. Analysis of several HNSCC cell lines and primary tumors has shown that transcriptional silencing through methylation of the 5′CpG island is one of the major mechanisms for P16 inactivation in this type of cancer [7].

The P15 gene, located 28 kb from the P16 gene, shares a high degree of structural and functional homology with the P16 gene. Functional studies have shown that P15 expression is upregulated by the transforming growth factor β (TGF β) [8]. Homozygous deletion, the most frequent alteration of the gene in many types of cancer, frequently occurs simultaneously with the deletion of P16. Expression of the P15 gene may also be controlled epigenetically by DNA methylation 9, 10. However, until now, reports concerning the status of P16 and P15 in MSCC have been nonexistant.

In the present study, the abnormalities of the P16 and P15 genes, including homozygous deletion, methylation of their 5′CpG islands, and mutations were investigated in 65 primary MSCC using MSP, PCR-SSCP, and DNA sequencing techniques.

Section snippets

Patients and Samples

Specimens of 65 primary maxillofacial squamous cell carcinomas and matched tumor-adjacent normal tissue were obtained from patients hospitalized at the Clinic of Stomatology, West China University of Medical Sciences. Of the 65 patients, 38 were males, 27 were females. They ranged in age from 30 to 77 years old, with an average age of 54 years. Lymph node metastases were detected in 23 cases. According to the criteria of the Union Internationale Contre Cancer (UICC), 13 cases were stage I, 26

Homozygous Deletion of the P16 and P15 Genes

Of the 65 tumors, none showed homozygous deletions in the P16 or P15 gene (Fig. 1). However, in some cases the

intensity of the amplified products of exon 1 in the P16 gene was weaker when compared to the normal controls.

Methylation Status of the P16 and P15 Genes

Studies on methylation status of the P16 gene in 65 patients with primary MSCC showed that 22 bisulfite-modified tumor DNA samples could be amplified not only with primers for unmethylated DNA (P16-U), but also by primers for methylated DNA (P16-M), while their matched normal

Discussion

Methylation is one of major mechanisms of gene regulation in the normal developmental process and tumorigenesis. Hypermethylation of 5′CpG islands of the P16 or P15 gene in some human cancer lines has been reported to be associated with a complete RNA transcriptional block 7, 8. Such hypermethylation has also been reported in many primary cancers, including cancers of the bladder, breast, prostate, colon and head and neck organs 14, 15, 16, 17, 18. However, studies of the status of P16 and P15

Acknowledgements

We would like to thank Doctors Lie Chen, Gang Luo and Minhai Nie for providing pathological materials, and help in immunohistochemical experiments. This work was supported by National Natural Science Foundation of China.

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