Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study
Introduction
Cervical cancer is the leading female cancer in Korea and the etiologic association between human papillomaviruses (HPVs) and cervical cancers has been established [1]. The E6 and E7 genes of oncogenic HPVs are known to play an important role in the process of malignant transformation and immortalization of cervical epithelial cells as initiators in the early stage [1], [2], [3]. Inactivation of p53 and Rb by E6 and E7 proteins from HPV-16/18 is an important process to maintain abnormal cellular proliferation by disturbing the normal cell cycle [3], [4], [5]. However, HPV infection alone is probably insufficient for complete neoplastic transformation of the cervical cells, suggesting the involvement of other genetic and epigenetic events in cervical carcinogenesis. The role as promoting factors in the later stage of progression to cervical malignancy remains uncertain. Previous cytogenetic studies have found the numerical and structural abnormalities of chromosome 9 in cervical cancers, suggesting the presence of putative tumor suppressor gene(s) on this chromosome [6], [7]. Alterations of the p16INK4 gene mapped on band p21 of chromosome 9 have been detected in various gynecologic malignancies, but the role of the gene as a tumor suppressor in vivo appears to be dependent on tumor type [8]. Recently, p16 protein is known to bind to cyclin dependent kinase 4 (CDK4), and inhibit the ability of CDK4 to interact with cyclin D and stimulate passage through the G1 phase of the cell cycle [9], [10]. Deletions or mutations in the p16 gene may affect the relative balance of functional p16 and cyclin D, resulting in abnormal cell growth [11], [12]. Loss of p16INK4 inhibition of the cdk4-cyclin D complex could result in constitutive phosphorylation of Rb and unregulated cell growth [13]. Several groups reported that the p16INK4 gene was homozygously deleted or mutated in many type of cancers [8], [9], [10], but the relatively low incidence of p16INK4 gene alterations detected in cervical cancers indicated that this abnormality might not be an important event in the pathogenesis of cervical cancer [14], [15]. To determine whether genetic changes in the p16 gene are involved in the pathogenesis of cervical cancer, we examined DNAs by LOH study and sequencing from squamous cell carcinomas and their adjacent dysplastic lesions using a precise tissue microdissection for detecting the presence of HPV-16/18 DNAs, the genomic changes at p16 and Rb genes.
Section snippets
Microdissection and DNA extraction
Thirty-four patients with squamous cell carcinoma of the uterine cervix who were treated at the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Kangnam St. Mary's Hospital, Catholic University Medical College, Seoul, South Korea, were included in this study. Dysplastic, carcinoma and adjacent normal cells were procured from formalin fixed, paraffin-embedded slides selectively obtained by microdissection with a 30-gauge needle (Becton Dickson, NJ) (Fig. 1), which was
Results
Of the 34 squamous cell carcinomas of uterine cervix, 24 cases (80%) had HPV-16 DNA and 2 cases had HPV-18 DNA (co-infected with HPV-16). Gene alterations of D9S171 and IFNA for 9p21-22 marker (p16INK4a) were detected in 18% (6/34) at one or more loci; 2 cases were from both dysplastic and cancer cells and 4 cases were only detectable from cancer cells. Two of 27 (7%) informative cases showed loss of heterozygosity, 2 cases showed homozygous deletion at D9S171, 2 of 19 (11%) showed loss of
Discussion
Uterine cervical carcinomas are preceded by precursor cervical intraepithelial lesions (CINs) and high-grade CINs have a much greater potential of progressing to cervical cancers than low-grade CINs [1], [2], [3]. The presence of specific HPV types and the integration of these HPV DNAs are considered the important factors in the process of cervical carcinogenesis and progression of the disease [1], [2], [3], [17], [18], [19]. The loss of E2 function by integration of HPV DNA can cause
Acknowledgements
This work was supported in part by HMP-97-M-2-0020 and in part by KOSEF through the Cancer Research Center at Seoul National University.
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