University of California, Berkeley, scientists have discovered the extremely subtle effect that the prescription drug Taxol has cells that it is one of the most widely used anticancer agents in the world.
The details, which the drug interference with the normal function of microtubules, part of the cell skeleton, could help in designing better drugs, or in improving Taxol and other drugs known to disturb the functioning of the microtubules.
The findings are reported in the journal cell.
"Efforts towards understanding this better chemotherapeutics are very important, because there are some differences microtubule in cancer cells versus normal cells that maybe we can exploit," said principle Author Eva Nogales, a biophysicist, UC Berkeley professor of molecular and cell biology and senior faculty scientist at Lawrence Berkeley National Laboratory (LBNL). "We are not there yet, but this is the kind of analysis we need to get there."
Taxol, originally extracted from the bark of the Pacific Hangul:, is one of the most commonly used medicines against solid tumors, and is a front-line drug for the treatment of ovarian cancer and advanced breast. The drug is known to bind to microtubules and freeze them essentially in place, which prevents the chromosomes to separate them when a cell divides. This will kill dividing line cells, particularly cancer cells, which are known for the rapid proliferation.
Nogales, a Howard Hughes Medical Institute researcher, has been working on microtubules since she was a doctoral student in England in the early 1990s, using techniques such as x-ray scattering and cryoelectron microscopy to study influence of Taxol and other anticancer agents on the microtubules. Later, during her postdoctoral work on LBNL with Ken Downing, she was the first to discover exactly where Taxol binds the basic building block, called tubulin, microtubule polymer.
Microtubules are of the cell skeleton
Work by many scientists around the world have shown the microtubule network within cells called the cytoskeleton, very different from stiff animal skeletons. Microtubules are polymer by amalgamation of filaments that are constantly growing and shrinking, and in doing so push and pull things around the cell, including the chromosomes. Scientists call this dynamic instability. The microtubules also offer a highway for the transport of the cell organelles and other packages around the cell.
Tubulin, microtubules, the basic structural unit is a complex of two proteins-alpha and beta tubulin. Tubulin units stack up a on top of another to align with other comics strips that form and then zip up to form a hollow tube, the microtubules.
"The cytoskeletal protein Tubulin in microtubules, assembles, that itself is absolutely essential to the life of every eukaryotic cell, which is why it has become a prime target of anticancer agents to," said Nogales. "It's amazing how microtubules probe and try new things almost random, but there is a level of control built into the cell that ultimately makes sense for this chaos, and the cell survives and flourishes."
Microtubules grow their free end about 1 micron per minute by continually adding more tubulin (about 20 tubulin molecules per second). But if they stop growing, they quickly peel apart as the skin of a banana, releasing tubulin for recycling in other microtubules. This peeling, or depolymerization, takes place on up to 15 micrometres per minute, or about 300 tubulin molecules falling off per second, Nogales said.
Microtubules are as compressed springs
Nogales has now discovered why microtubules as quickly peel apart. When they assemble, are put under intense pressure, but the strips tubulin bending and pulling apart prevented by the growing cap of tubulin in the end. Once growing stops and that Cap disappears, rips the understated tension the microtubule from each other.
The tension is created when the tubulin complex, which has a small energy molecule called GTP (Guanosine triphosphate) attached, and the GTP is hydrolysed in GDP ((III) calcium Guanosine diphosphate changes). This chemical reaction encrypts, compresses the alpha and beta subunits, much like compacted vertebrae, keep the tubulin stack under tension, as long as the microtubule on its end is growing.
"It had proposed that tubulin was to be limited, but nobody had it turned out to be," said Nogales. "What we have seen is that if GTP hydrolysis occurs, the tubulin structure gets stuck in a tense situation, like a compressed spring. The subunits end are to hold the whole thing together. "
If growth stops, the tension is released, and quickly Peel the strips apart.
"This work represents an important step forward on an issue with a long history," wrote Tim Mitchison in a commentary in the same issue of cell. Mitchison, a professor of Harvard University in systems biology, was the first to recognize the importance of GTP hydrolysis in destabilizing microtubules. The model proposed by Nogales and her team, he added, "offers our first glimpse in (the) mechanism destabilization."
Nogales also found that Taxol itself is inserted into the protein tubulin and prevent the compaction of the alpha and beta subunits, so no excitement builds. As a result, even if the microtubule stops growing, remains intact, basically frozen in place, peel apart, or depolymerize, and its normal function.
"Taxol reverses the effects of GTP hydrolysis," she said.
Nogales and her team discovered these structural changes by pushing the limits of cryoelectron microscopy, a technique in which samples are frozen and probed with a high-powered electron beam. They now have a resolution sufficient to details smaller than 5 angstroms (a tenth of a nanometer), which about the size of five hydrogen atoms is reached. While most information to date about the structure of tubulin in the microtubule has come from the study of artificial, flat sheets of aligned strips tubulin, Nogales was able to probe three dimensional microtubules frozen in their natural state, with and without Taxol bound to tubulin. This comparison showed clearly the effect that taxol has on microtubule structure.
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