|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
“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).
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