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Academician G.P. GEORGIYEV, Director of the Institute of Gene Biology, USSR Academy of Sciences
Despite frequent reports on the discovery of effective drugs for combating malignant tumors, cancer is still the leading killer disease. Therefore we must point out that the work discussed here, namely, the discovery of a metastasine gene, is just a step towards the discovery of the molecular nature of cancer, the disease of man's genome, the heredity apparatus of the cell.
The work has been done by our laboratory's group headed by Ye. Lukanidin, Dr. Sc. (Biol.). The group has now become an independent laboratory in the newly founded Institute of Gene Biology of the USSR Academy of Sciences. Its staff deals with the molecular principle of change in the heredity system during the appearance and development of tumors. The group has been trying for years to discover the mechanics of metastasis, for example, to use the DNA to turn metastasing cells into unmetastasing ones, and vice versa. However, all attempts failed.
The researchers then turned to another field. The idea was to isolate a gene responsible for the regulation of metastasis. To do so, they took two lines of the cancerous cells of mouse's milk gland of the same origin, that is, obtained from the same maternal line. One line did not produce metastases at all or produced them very seldom, in the late stages only The other produced metastases in the lungs and lymph nodes steadily and with a very high frequency-something was happening in the cancerous cells. And the latter were selected as models.
Man's genome consists of 3 bln nucleotide pairs, (kindred, but different chemical combinations), and each of 10 cells making up the human organism contains this amount of nucleotides. All the properties of the human organism are recorded in its genome. As for cancer, it is now viewed as the disease of man's genome.
A search has got under way for genes which worked in the metastasing line of cells, gave out information causing a synthesis of proteins and did not do so in the non-metastasing line.
Without going into details, let us explain that neither the work of genes (the matrix or sections of the DNA) nor the synthesis of protein is possible without ribonucleic acids-RNA; if the gene works, a synthesis of the matrix RNA (mRNA) takes place on it. We must also note the gene should be obtained in the shape of the DNA formula since RNA molecules cannot be multiplied.
The researchers took the matrix RNA of the metastasing line and synthesized the corresponding (complementary) DNA with the aid of a special ferment, revertasis. Whereupon they made the so- called subtraction, in other words, connected the synthesized DNA with the mRNA from the non-metastasing line. Everything that was connected or hybridized was rejected: the DNA molecules identify for the metastasing and non-metastasing cells "recognized" one another, linked among themselves, and withdrew from the reaction.
The molecules that failed to find partners - that is what makes one line of cells different from another - were used for further research. That was what the process of subtraction consisted in. What was left after it was the DNA corresponding to the mRNA. Such DNA are present in the metastasing line and absent in the other.
The next job was to multiply or, as is customary to say, to make copies of these molecules with the aid of the genetic engineering methods. The remaining unhybridized DNA were built into the DNA of the viruses of bacteria (they are called phages) to obtain clones- bacteria bearing the heredity material of the changed phages. The clones was later copied and the DNA isolated from them was hybridized with the complementary mRNA of the metastasing and non-metastasing lines. That was followed by the selection of the clones which hybridized with the mRNA of metastasing cells and did not hybridize with that of the mRNA of the non-metastasing ones.
In the long run the researchers discovered a clone containing a gene present in the metastasing tumor and absent in the non- metastasing one. It signified that the gene was working in the given line of the metastasing cells.
A more thorough checkup was followed to test a series of metastasing and non-metastasing tumors of many different
lines of cancer. The work was done together with specialists from the All-Union Cancer Center of the USSR Academy of Medical Sciences. The researchers isolated the RNA from several different tumors in mice and studied hybridization with the obtained gene. It turned out that the gene worked in all the metastasing tumors for a very few exceptions and did not work in the non-metastasing ones. Besides, it was found that it did not function in most of the types of normal cells, but rather actively multiplied in those responsible for immunity.
After it was shown that the gene worked only in many metastasing cells, it was called metastasine-1 (mts-1).
The next stage was to decipher the primary structure of the gene which coded its protein product. A computerized structural analysis of the protein revealed that there was pure analog in the data bank, that is, it turned out that a fundamentally new gene was obtained. However, a degree of similarity with other proteins was found nevertheless. It appeared that the protein, the product of the mts-1 gene, belonged to a large family of cell proteins capable of binding calcium. True, this should not be taken to mean that this fact explains its inclination to form metastases although such proteins may play a regulatory role in the life of the cell.
It was vital to find out to what extent the mts-1 determined the metastasing process. This question could be answered by an attempt to turn the non-metastasing cells into metastasing ones with the aid of the gene. M. Grigoryan, a researcher from Lukanidin's group, went to see their American counterparts at Rochester University where experiments were made to specify the biological role of the mts-1. The researchers introduced structures containing an actively working mts-1 into non-metastasing cells where the mts-1 did not work, that is, infected them, as it were. If such a structure was introduced into a cell genome and worked actively, the tumors in this cell acquired the ability to metastase.
In the meantime, similar experiments were made in Moscow where the same gene was used, but in a reverse and not straight orientation. With the aid of a special ferment it was read in an opposite direction to obtain the inverse sequence of the gene. When this structure actively worked in metastasing cells, the inverse sequences being synthesized were bound with the positive (correct) mRNA to which they corresponded and switched it off. In fact, the gene switched itself off. And the cell drastically reduced its ability to form metastases.
More and more facts are being accumulated about a direct connection between the work of the mts-1 gene and the ability of tumor cells to develop metastases. Nevertheless, it is too early to draw final conclusions, what is needed are additional experiments.
Meanwhile, M. Grigoryan carried out new research in Rochester. She studied the protein product of the mts-1 gene, that is, a protein called metastasine. Since the structure of the protein was known-it directly followed from the structure of the mts-1 gene-polypeptides, sections of the protein chain were synthesized. They were used, already in Moscow, to obtain antibodies capable of linking up both with peptides and the metastasine of mice. Consequently, the way was opened to a large-scale study of metastasine, its contents in tissues, its distribution inside the cell and its participation in cell metabolism.
This work, accomplished by Lukanidin and Grigoryan, is currently being carried on in Moscow. The first results have been obtained, making it possible to determine the place of metastasine in the cytoplasm of metastasing tumor cells.
After the gene of metastasine (mts-1) was isolated from the cells of mice and its structure was discovered, Lukanidin's group turned to man's genome. The gene of man's metastasine was isolated and described. It is close to, although not identical with a mouse gene. To study the wide spectrum of human tumors, A. Ebralidze, another associate of Lukanidin, went to the Norwegian Cancer Study Center having a large collection of man's metastasing and non-metastasing cellular lines. The idea was to find out how mts-1 worked. To this end, he isolated a matrix RNA (mRNA), divided it according to the molecular weight with the aid of electrophoresis, and carried it over to filters where it hybridized with the labelled (tagged) gene of man's metastasine. It became clear that if RNA synthesized on mts-1 are present in the mRNA preparation, they bind the tagged DNA of the mts-1 gene. An X-ray film, when a filter is imposed on it, shows an exposed strip, and one can exactly determine the intensity of the gene's work by it. The brighter the strip, the more actively the gene works. A comparison revealed that for different tumors in man existed a direct interconnection between the intensity of the mts-1 work and the degree of the malignancy of the tumor - metastasing.
Ye. Tulchinskiy, also of Lukanidin's group, has now begun to study the regulation of the mts-1 work. A search is under way for the sections of the DNA responsible for the gene's active work in metastasing cells.
We might say that the gene consists of two parts, regulatory and structural. Two spheres, one before the gene, the other inside it, have been found. They are necessary for the effective work of mts- 1. It is known that proteins determining the RNA synthesis are bound with such DNA regulatory sections. The problem is to isolate those proteins and the genes coding them which would make it possible to read their heredity information. As a result, we will have genes governing the work of mts-1.
In the meantime, scientists are also searching for new genes linked with metastasing. Another gene, to be more exact a part of the gene, is now known. It was selected the way the gene of metastasine had been chosen, but only from other tumors. It was isolated, and cloned, and its primary sequence is being analyzed. Later this sequence will be let through the computer data bank to see if is known. If it is not, the problem will be to find out why the particular gene works chiefly in metastasing cell and how it is connected with metastasine. It is a new chapter.
Cooperation proposals are now coming to the Institute laboratories from many countries. Ye. Tulchinskiy is going to Rochester to continue to study the regulation of the work of the metastasine gene. Joint research is underway with colleagues in Norway. They were joined by scientists from France a short while ago.
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