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Isolation of a Cervical Cancer Tumor Suppressor Gene

Principal Investigator
Srivatsan, Eri Ph.D.

Contact Phone Number
(310) 268-3106

Director Bio

Dr. Srivatsan earned his doctoral degree in biochemistry from the Indian Institute of Science in Bangalore, India in 1977. He served as a postdoctoral fellow in the Department of Pathology at UCLA from 1977 - 1979 followed by two years at UC San Diego in the Department of Pediatrics. Dr. Srivatsan then served as a Research Specialist in the Department of Microbiology and Molecular Genetics at UC Irvine and as Research Assistant Professor in the Department of Pediatrics at the Children. s Hospital of Los Angeles. He joined the UCLA faculty in the Department of Surgery in 1994. Dr. Srivatsan heads the molecular biology laboratory at the West Los Angeles VA Medical Center. His primary research interests include the localization and isolation of human tumor suppressor genes and physical linkage mapping of specific chromosomal regions.


Cervical carcinoma is one of the leading causes of cancer death in women worldwide with approximately 500,000 deaths per year. The isolation of the cervical carcinoma gene would lead to an improved understanding of the pathogenesis of these tumors. This, in turn, would lead to improvements in the diagnosis, treatment, and prognostic evaluation of these patients. Additionally, chromosome 11q13 is also implicated in the development of breast cancer, head and neck tumors, and in MEN1 tumors. A candidate gene for diabetes is also localized to chromosome 11q13. The cervical gene, once isolated, can be used to determine whether the gene plays a role in any of these human diseases.

Introduction

Annually, cervical cancer affects an estimated 420,000 women worldwide and 15,000 in the United States. Although human papilloma viruses have been implicated in the etiology of the disease, it has been shown that viral infection is not sufficient to induce neoplastic transformation of the cervical epithelial cells. Additional genetic factors such as activation of oncogenes or inactivation of tumor suppressor genes may be required. Studies of oncogenes have not shown a clear involvement of these genes in this tumor. Of the tumor suppressor genes, mutations of the Rb and p53 genes have been detected, implicating these two genes in tumor development, and it seems that these two genes may play a role in progression of the disease. This laboratory is interested in identifying genetic markers that may be involved in early stages of the disease.

Research

The cervical carcinoma cell line HeLa was used as a model system to identify tumor suppressor gene(s). Tumorigenic studies were performed by injection of the cells into immunologically suppressed nude mice. Somatic cell fusion of the tumorigenic HeLa cells with non-tumorigenic fibroblasts resulted in the suppression of the tumorigenic phenotype. Rare tumorigenic segregants, that had lost chromosomes, were isolated, indicating loss of tumor suppressor genes localized to these chromosomes. Genetic analysis of the non-tumorigenic and tumorigenic hybrids showed the gene(s) to be localized to normal chromosome 11s. These results were confirmed by single chromosome transfer studies, wherein a single chromosome 11 was introduced into the HeLa cells and shown to result in tumor suppression. Extensive molecular genetic studies, performed on the hybrids derived from single chromosome 11 transfer experiments, localized the gene to the q13 region of chromosome 11. This region encompasses 10 Mb of DNA that could code for at least 200 genes. Long range restriction mapping studies were undertaken on HPV positive and HPV negative cervical cell lines which further reduced this distance to 3 Mb. Physical mapping of markers from this 11Q13 area, carried out in this laboratory and others, have linked this 3 Mb distance in 40-100 kb genome lengths cloned in bacteria and yeast. It should now be possible to use these cloned fragments for the analysis of the non-tumor and tumor cells to isolate the 11q13 tumor suppressor gene.

The important step in the isolation of a tumor suppressor gene is to identify mutations of the gene in the tumor cells. The mutations include large genomic deletions or single nucleotide point mutations. Genomic deletions can be observed by hybridization or PCR analysis of the tumor cells with genetic markers localized to the 3 Mb area of 11q13. This analysis has shown homozygous loss (complete deletion) of a 220bp sequence (observed as a loss of PCR product) in the tumorigenic HeLa cells. The boundary of this deletion was narrowed to a 100 kb distance using additional markers on either side of the deleted segment. This indicates that the gene is localized within this 100 kb distance. In order to isolate all the genes coded by this area, the markers are linked in sub-genomic fragments of 25-40 kb. The individual sub-genomic fragments are used for the precise identification of the deletion breakpoints, i.e. sequences of the tumor suppressor gene. The genomic fragment can then be used to isolate cDNA (functional part of the gene), which when sequenced, will be utilized for the identification of mutations in the primary tumors. The homozygous deletion of the 11q13 marker was also observed in two of the HeLa cell derived tumorigenic hybrids, and in two of twenty primary tumors. Homozygous deletion is a rare event and involves deletion of a small segment of the genome. These deletions represent the location of tumor suppressor genes as evidenced by the isolation of Rb, DCC, P16 and DPC4 genes from marker regions homozygously deleted in retinoblastoma, colon cancer, melanoma and pancreatic cancer, respectively. Thus, hopefully, studies will result in the isolation of an important tumor suppressor gene localized to chromosome 11q13.

Significance

The important step in the isolation of a tumor suppressor gene is to identify mutations of the gene in the tumor cells. The mutations include large genomic deletions or single nucleotide point mutations. Genomic deletions can be observed by hybridization or PCR analysis of the tumor cells with genetic markers localized to the 3 Mb area of 11q13. This analysis has shown homozygous loss (complete deletion) of a 220bp sequence (observed as a loss of PCR product) in the tumorigenic HeLa cells. The boundary of this deletion was narrowed to a 100 kb distance using additional markers on either side of the deleted segment. This indicates that the gene is localized within this 100 kb distance. In order to isolate all the genes coded by this area, the markers are linked in sub-genomic fragments of 25-40 kb. The individual sub-genomic fragments are used for the precise identification of the deletion breakpoints, i.e. sequences of the tumor suppressor gene. The genomic fragment can then be used to isolate cDNA (functional part of the gene), which when sequenced, will be utilized for the identification of mutations in the primary tumors. The homozygous deletion of the 11q13 marker was also observed in two of the HeLa cell derived tumorigenic hybrids, and in two of twenty primary tumors. Homozygous deletion is a rare event and involves deletion of a small segment of the genome. These deletions represent the location of tumor suppressor genes as evidenced by the isolation of Rb, DCC, P16 and DPC4 genes from marker regions homozygously deleted in retinoblastoma, colon cancer, melanoma and pancreatic cancer, respectively. Thus, hopefully, studies will result in the isolation of an important tumor suppressor gene localized to chromosome 11q13.
 

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