Oct. 7, 2003 COLUMBUS, Ohio – A pattern produced by a chemical change that turns off genes in tumor cells may help predict the seriousness of a particular cancer, and perhaps its outcome.
The study by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute examined how a chemical change known as methylation spreads from one region of a breast-cancer gene to a neighboring region in tumor cells taken from patients.
The findings provide insight into how the methylation process progresses until it inactivates genes such as tumor suppressor genes that otherwise help protect against cancer. The findings are published in the Oct. 1 issue of the journal Cancer Research.
"Methylation is as important as gene mutations and chromosomal damage in the cancer process," says principal investigator Tim Hui-Ming Huang, associate professor of human cancer genetics.
"Our findings suggest that the degree of methylation may correlate with the seriousness of the tumor. If that proves to be true, it would have important implications for cancer diagnosis and predicting a patient's prognosis."
Methylation is the addition of small chemical units known as methyl groups to DNA. Cells normally use methylation to inactivate unneeded genes during embryonic development and throughout life. Abnormal methylation, however, occurs in many types of cancers.
The investigators developed microarray technology to measure methylation levels along two regions of a gene known as RASSF1A, which becomes highly methylated in many kinds of cancer. Microarray technology allows researchers to measure changes in genes from many different tumors simultaneously.
The researchers examined the methylation profiles of RASSF1A genes taken from 37 primary breast tumors, seven breast-cancer cell lines and 10 samples of normal breast tissue.
Specifically, they measured methylation levels at 19 sites spanning two adjoining regions of the gene: the promoter region, which regulates the activity of the gene, and the neighboring first exon. Exons are stretches of DNA that contain the information for the protein described by the gene.
Genes from the normal breast cells showed low to moderate methylation in the exon and little or no methylation in the promoter. Nearly one-third of breast tumors sampled also showed low levels of methylation in the promoter region.
The remaining tumor cells and all the breast-cancer cell lines, however, showed moderate to high levels of methylation in the exon and various degrees of methylation in the promoter.
High levels of promoter methylation correlate with an altered structure of the gene, resulting in a tightly closed DNA configuration that prevents gene activation.
"Our findings show that progressive methylation occurs in tumors from patients and support the idea that methylation begins in the exons and extends into the promoter," Huang says.
Huang and his colleagues are now working to correlate methylation patterns in leukemia and ovarian and lung cancer with the behavior and severity of the disease. Grants from the National Cancer Institute supported this research.
(Note: Huang and colleagues also have the following paper in the same issue of Cancer Research.)
NEW METHOD SHOWS METHYLATION REQUIRES TWO ENZYMES
COLUMBUS, Ohio – Two enzymes known as methytransferases work together to regulate the methylation process, according to a study led by Tim Hui-Ming Huang, associate professor of human cancer genetics at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute.
The study appears in the Oct. 1 issue of the journal Cancer Research.
Huang and his colleagues used a line of laboratory-grown ovarian-cancer cells and a new technique known as small interfering RNA (siRNA). The study showed that blocking the two enzymes simultaneously resulted in a loss of methylation of three genes known as TWIST, RASSF1A and HIN-1 that were inactive due to methylation.
siRNA involves introducing into cells small pieces of RNA that cause the destruction of messenger RNA for a particular protein. Messenger RNA is a copy of a gene that determines the amino acid sequence of a protein. Blocking or destroying messenger RNA prevents a protein from being produced by a cell. This enables researchers to study how loss of particular proteins affects cancer cells.
The study further showed that following the loss of methylation, the three genes were re-activated, as measured by the levels of messenger RNA for each gene. Loss of methylation led to a seven-fold increase in the levels of TWIST and HIN-1 messenger RNA and a 15-fold increase in RASSF1A messenger RNA relative to controls.
"These findings show how small interfering RNA can be used for dissecting the molecular mechanisms of cancer, and its potential use for blocking cancer-related proteins as means of cancer therapy," Huang says.
This study was funded by grants from the National Cancer Institute.
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