Diagnose cancer by clones
Marina Muravyeva, STRF.ru
In most cases, cancer is detected only at a late stage, when it is almost impossible to save the patient. The Laboratory of Viral RNA Biochemistry at the Institute of Protein is developing technologies for early diagnosis. Moreover, in the future they can be used for the initial examination of the entire population or certain groups (age and professional) with an increased predisposition to cancer. For example, it would be advisable for employees of chemical enterprises to undergo special cancer testing as part of a routine medical examination. If a patient's blood sample (say, one milliliter) contains at least one cancer cell, one marker molecule, the disease will be detected. At the same time, the diagnosis will take only a few hours.
The existence of such sensitive test systems is still hard to believe. But scientists are already close to introducing their developments into clinical practice. New diagnostic technologies are based on the method of molecular cloning, the effectiveness of which has already been proven experimentally.
About clones growing in gelThe discovery of the method of molecular cloning (reproduction) of nucleic acids is one of the main achievements of the Laboratory of Viral RNA biochemistry under the leadership of Corresponding Member of the Russian Academy of Sciences Alexander Chetverin.
The method was developed in the late 1980s, just a few years after the creation of the laboratory (then still a scientific group).
"We assumed that RNA would multiply in the form of colonies: its offspring would appear in place of each initial molecule,– says Alexander Chetverin. – And so it turned out. Experimentally, we have confirmed our hypothesis."
The scientists chose polymerase chain reaction (PCR) as the main reproduction system. This experimental method, widely used in biological and practical medicine, makes it possible to significantly increase the small concentrations of certain fragments of nucleic acid. Unlike traditional PCR, which is carried out in a liquid, the laboratory of viral RNA biochemistry decided to carry out the reaction in a gel. And they achieved an important result: the offspring of each initial molecule did not fill the entire aqueous medium with itself, but remained in its cell, or pore in the gel, concentrating around the parent molecule. In other words, there was a colony of up to a billion molecules, each of which was a clone of the parent.
"The spatial separation of the cloned molecules is a unique feature of our method, which distinguishes it favorably from other methods of nucleic acid reproduction," says the head of the Laboratory of Viral RNA Biochemistry. "By eliminating competition between different types of molecules, we were able to register and count single molecules capable of reproduction, as well as analyze a huge number of molecules fairly quickly – thousands of colonies formed by them can be distinguished on one gel."
In the fight against molecular competitorsScientists from the Laboratory of Viral RNA biochemistry have already used the method of molecular cloning for the diagnosis of infectious diseases, in particular, they tested the new system on the example of AIDS virus RNA and hepatitis B virus DNA.
It has been experimentally proven that the sensitivity of the cloning method is much higher than that of standard methods based on the reproduction of RNA or DNA in liquid. Their main problem is the competition between the reproduction of the analyzed target (specific RNA or DNA) and non–specific molecules other than it.
"A clinical blood sample contains a huge amount of non–specific DNA, the mass of which is trillions of times larger than the target being determined," explains Alexander Chetverin. – Both molecules multiply at the same time. And it's not scary if there are a lot of targets initially – it will still be possible to detect it. But if there is not enough of it (for example, only one molecule), then the synthesis of competing molecules “clogs” the molecules of interest to us. So it will not be possible to detect the disease if you follow the traditional scheme of the reaction – in a liquid."
During the formation of colonies in the gel, the reproduction of specific targets and non-specific molecules occurs in different pores of the gel: occupying strictly their "ecological niches", they do not interfere with each other. In experiments, scientists from the Laboratory of Viral RNA biochemistry have shown that they are able to detect disease markers regardless of the size of colonies, their number and the excess of "competitive" nucleic acid molecules. If using traditional methods, from 100 to 1000 target molecules are needed for diagnosis, then with molecular cloning – only one or two. That is, this diagnostic system is several orders of magnitude more sensitive. Moreover, the technique is universal and can be used to diagnose any diseases for which there are RNA or DNA markers. Having successfully tested the method on infectious diseases (AIDS and hepatitis B), scientists decided to create test systems for cancer.
Universal cancer markersCurrently, the laboratory of viral RNA biochemistry is working on creating a system for diagnosing oncological diseases.
The research is carried out within the framework of the Federal Target Program "Development of RNA markers for the molecular diagnosis of cancer in digital format".
Scientists are going to diagnose this disease by detecting RNA molecules in clinical blood samples that are present in all cancer cells without exception. An example of such a universal marker is the matrix RNA (mRNA) of the telomerase protein, an enzyme responsible for the synthesis of the end sections of chromosomes (telomeres). This mRNA also occurs in normal stem cells, which, like cancer cells, are capable of unlimited division, are only in their niches and do not spread throughout the body. Thus, the presence of telomerase mRNA where there should be no stem cells (for example, in blood plasma) may indicate the presence of a malignant process.
"Our task is to determine a list of similar marker RNAs that could be recommended for large–scale clinical trials," says Alexander Chetverin. – I think these tests will be available to a wide range of people. They differ from the usual liquid PCR test only in that they are made in a gel, and not in a test tube. So the cost will also be comparable – in the region of several thousand rubles. Although if they are used en masse, the price can be reduced to 300-500 rubles."
The test guarantees a fairly fast result: it takes from half an hour to several hours to isolate nucleic acids from a blood sample, then it will take about two more hours to carry out the reaction itself.
Scientists expect that they will be able to create an absolutely sensitive diagnostic system. "I can't be an oracle, but based on previous experience, we have every reason to hope for the creation of such a technology," says Alexander Chetverin. – If we have all the necessary control tests, we will be able to say for sure whether a person has cancer or not. The latter, by the way, is also important. There are a lot of examples of false diagnosis, because of which people's lives break down, often it comes to suicide. And then suddenly it turns out that the doctors made a mistake: the defect of the film on the X-ray was mistaken for a tumor. It is also necessary to save people from this – not only from cancer, but also from making an incorrect diagnosis."
Become the first, as with a flight into SpaceThe technologies created in the laboratory of viral RNA biochemistry have no analogues in the world.
But the prospects for their implementation still look vague: there is no corresponding base and specialists at the academic institute for the production of the necessary devices and reagent kits. Alexander Chetverin and his colleagues have already tried to implement their developments, so far to no avail. With a proposal for cooperation, they, for example, applied to the Institute of Epidemiology, where they are engaged in the diagnosis of AIDS. Interest was not met. Some companies would like to undertake this project, but do not have the necessary budget.
"It will be very disappointing if our invention is the first to be used in the West, and then we will buy either devices or licenses for their production from foreign companies," says Alexander Chetverin. – Now we have an absolute priority. This is one of the rare areas where you can really make a breakthrough. Once upon a time, the Russians were the first to send a man into Space. It's the same here: you can be the first to create a unique diagnostic system."
The situation can get off the ground if all the necessary specialists, including production workers, are involved in the project. Despite the fact that large investments are needed, Alexander Chetverin is confident that all costs will be compensated. The developed technique is universal and can be used not only in medicine, but also in agriculture, criminology and many other fields of activity.
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05.05.2008