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Thursday, December 21, 2006
Researchers Find That a ‘Silent’ Gene Mutation Can Change the Function of an Anticancer Drug Pump
A genetic mutation that does not cause a change in the amino acid sequence of the resulting protein can still alter the protein’s expected function, according to a new study conducted at the National Cancer Institute (NCI), part of the National Institutes of Health (NIH). The study shows that mutations involving only single chemical bases in a gene known as the multidrug resistance gene (MDR1) that do not affect the protein sequence of the MDR1 gene product can still alter the protein’s ability to bind certain drugs. Changes in drug binding may ultimately affect the response to treatment with many types of drugs, including those used in chemotherapy. The results of this study appear online in Science Express on December 21, 2006*.
The genetic mutations examined in this research are known as single nucleotide polymorphisms (SNPs) and are very common. Some SNPs do not change the DNA’s coding sequence, so these types of so-called ‘silent’ mutations were not thought to change the function of the resulting proteins.
“This study provides an exception to the silent SNP paradigm by showing that some minor mutations can profoundly affect normal cell activity,” said NCI Director John E. Niederhuber, M.D. “These results may not only change our thinking about mechanisms of drug resistance, but may also cause us to reassess our whole understanding of SNPs in general, and what role they play in disease.”
Despite success in treating some cancers with chemotherapy, many tumors are naturally resistant to anticancer drugs or become resistant to chemotherapy after many rounds of treatment. Researchers at NCI and elsewhere have discovered one way that cancer cells become resistant to anticancer drugs: they expel drug molecules using pumps embedded in the cellular membrane. One of these pumps, called P-glycoprotein (P-gp), is the protein product of the MDR1 gene and contributes to drug resistance in about 50 percent of human cancers. P-gp prevents the accumulation of powerful anticancer drugs, such as etoposide and Taxol, in tumor cells. The same pump is also involved in determining how many different drugs, including anticancer drugs, are taken up or expelled from the cell.
In this study, researchers led by Michael M. Gottesman, M.D., head of the Laboratory of Cell Biology within NCI's Center for Cancer Research, demonstrated that SNPs in the MDR1 gene result in a pump with an altered ability to interact with certain drugs and pump inhibitor molecules. In order to show that SNPs can actually affect pump activity, the researchers genetically engineered cells in the laboratory to contain either normal MDR1 or a copy of the MDR1 gene that contains one or more SNPs. Then, they used fluorescent dyes to track pump function based on the accumulation of dye in the cell or the export of dye out of the cell with and without various inhibitors of P-gp. This showed that although the SNPs did not change the expected P-gp protein sequence, the presence of one particular SNP, when in combination with one or two other SNPs that frequently occur with it, resulted in less effective pump activity for some drugs than normal P-gp without the SNP.
The P-gp protein sequences and production levels were normal in both the cells that received the normal MDR1 gene and those that received the mutant versions. Therefore, in order to determine how the SNPs affected pump function, Chava Kimchi-Sarfaty, Ph.D., lead author of the study, and co-workers used an antibody that could distinguish between different P-gp structural conformations. They found significant differences in antibody binding consistent with the existence of different protein conformations in the products of MDR1 genes with or without the SNPs. These results indicate that the shape of a protein is determined by more than its amino acid — or primary — sequence.
Like all proteins, P-gp is comprised of amino acid building blocks. While making P-gp, the cell’s protein synthesizing machinery knows exactly which amino acids to put together and in which order by reading a copy of the MDR1 gene coding sequence. DNA consists of a sequence of chemical bases, and the code for individual amino acids is represented by specific sets of three adjacent DNA bases called codons. The SNP that Gottesman and his colleagues studied had only one changed base in one codon of the MDR1 gene. Since several different codons can contain the code for the same amino acid, this SNP only altered the gene by converting one common codon to a rare one, but did not change the amino acid for which it coded.
“We think that this SNP affected protein function because it forced the cell to read a different DNA codon than it usually does,” said Gottesman. “While the same exact protein sequence eventually got made, this slight change might slow the folding rhythm, resulting in an altered protein conformation, which in turn affects function.”
Since silent SNPs are frequently found in nature, their biological role has largely been overlooked. However, this study raises the possibility that even ‘silent’ mutations could contribute to the development of cancer and many other diseases.
For more information on Dr. Gottesman’s research, go to http://ccr.nci.nih.gov/staff/staff.asp?profileid=5713.
For more information about cancer, please visit the NCI Web site at http://www.cancer.gov, or call NCI's Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).
About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.
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* Kimchi-Sarfaty C, Mi Oh J, Kim I-W, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM. A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science Express, December 21, 2006.