Abstract
Brief description of scientific life of Russian biochemist G. I. Abelev in tumor immunochemistry. Main attention is devoted to develop the system of analysis of complex system on the level of individual antigens. The discovery of alpha-fetoprotein and its use as tumor marker is described.
In my childhood and youth I knew nothing about the existence of research institutes and researchers. No one among the members of my family or their friends was involved in research. My interest in science arose in the time of World War II when I was a schoolboy. In the very beginning it was mainly of philosophical type. I wanted to understand the thinking process and how everything is organized. Perhaps initially I was interested in astronomy, particularly due to the popular books by Flammarion.
My school years – from the 6th to 10th classes – proceeded in the wartime. Naturally, life at that time was very hard for everybody, and the harder it was, the more attractive seemed to me the life in science – bright, interesting, and noble. Life within the walls of university became the goal of all my dreams. After the 8th class I acquired more definite interest in psycho-logical problems. I tried to read Ivan Sechenov, Vladimir Bekhterev, Ivan Pavlov, and I even organized a home «seminar» where we discussed their studies and problems related to psychology.
Graduating from school in 1945, I was at the «crossroad»: what institute should I try to enter? At that time the Chair of psychology had just been opened at the Department of Philosophy, Moscow State University. I had no interest in biology in itself, but I was interested in physiology of higher nervous activity and of the work of the brain.
At that time I used to listen to public lectures in the Moscow Polytechnic Museum. The distinguished physiologist Professor P. K. Anokhin had given a lecture on higher nervous activity. Although I could not dare to ask him for advice, I felt that he was the person who may really help me to choose my own way. At last I became brave enough to ask his advice, saying that I am particularly interested in what are the brain and the thoughts? Anokhin gave me a clear answer. He said that if you enter the Chair of Psychology it will be interesting for you to learn and read literature, but nothing would be done by yourself. Studying physiology you cannot do much, but all you will have done would be your own work. His advice was consistent with my own ideas and desires, and so I went in for the entrance examinations at the Department of Biology, Moscow State University. Although I prepared for these exams very seriously, I nearly failed. The reason was that my entrance to the university meant too much to me and I probably overworked during preparation for the examinations.
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| With father – Israel Efimovich Abelev and elder brother Samuil (1938) | Mother – Evgenia Samuilovna Abeleva (Maiden name Gorfinkel) |
The competition for entrance was quite strong, about six competitors per university seat. In spite of my so-called semi-acceptable score, I was accepted as a student of the Biological Department, possibly due to my gender. At that time the majority of boys preferred to enter Moscow Aviation Institute, Moscow Energetic Institute, Moscow Technical High School, or at least the Departments of Physics or Mathematics of Moscow State University.
University
Soon after my entrance to the Biological Faculty, I became a member of the Students' Scientific Society at the Chair of Physiology of Higher Nervous Activity. It was directed by Professor Kh. S. Kashtoyants and Associate Professor M. V. Kirzon. Possibly due to my activity (I was interested in this subject) Kashtoyants said to his students that he would like to talk with me.
However, I was very shy and did not see him. Soon after this he suddenly died. With my friend Alexander Zotin, who later became Professor and Head of the Laboratory at the Kol'tsov Institute of Developmental Biology, we organized our own student scientific society. We called it «student scientific society group for biophysics», but in reality we were interested in mechanisms determining physiological processes. Now this field of science is known as molecular biology. We invited Professor S. S. Vassiliev, who gave lectures in physical chemistry at our Biological Faculty, to become the head of this group. The personality of this enthusiastic charming man substantial influenced my life and those of my friends. Zotin worked on energetics of development all his life, and his research interests originated from problems that we analyzed with Professor Vassiliev in our student scientific society group.
During the first and second years, with 3–4 other students, began to work with Professor Alexander Gurvich, an outstanding world-renowned cell biologist, who proposed the theory of biological field. (He used to be the Director of the Institute of Experimental Biology of the All-Union Institute of Experimental medicine.) We visited his laboratory after lectures and seminars (and sometimes instead of them!) and worked there until very late in the evening. This was our first involvement in real research work. It was neither simple nor easy.
Thus, from the very beginning of my studentship, I met and worked with outstanding people in remarkable laboratories and institutes. I think that Moscow State University of the postwar period (up to the sadly known August 1948, when Lyssenko came to power in biology) had major influence on my formation as a scientist. I should say that even the university walls, painted by famous Russian artist Vasnetsov, «emitted» special aura and charm.
Romantics, moral purity, democratic principles, nice people, and real scientists open to communication with students created my image of science, type of relations and communications, and the norm of scientific activity which remain with me all my life.
Among professors who influenced my choice of scientific interests and my way in science, I should again mention S. S. Vassiliev, the head of our scientific student group. He worked with us, his students, regretting neither his time nor effort. Especially for us, a small group of students, he prepared wonderful courses of lectures on mathematics, physical chemistry, chemical kinetics, and thermodynamics. This experience, together with knowledge I gained from his lessons, still remains with me, and I am very grateful to Professor Vassiliev.
Professor Alexander Gurvich and his daughter, Anna Gurvich, with whom I worked during the first and second years of my studentship, had no direct relation to Moscow State University. Nevertheless, they significantly influenced my viewpoints on science and scientific human relations. I accepted their type of human relations, scientific spirit, and democracy. We «inhaled» their aura, which still remains inside us as some scientific norm, requested type, and mode of scientific relations.
In Gurvich's laboratory I was working on the problem of the nature of nerve excitation, which attracted my interest and «drove» me to this particular laboratory. I thought that I could get inside elemental steps of the nerve process. In agreement with Gurvich's theory, I thought that using mitogenetic emission* accompanying the process of nerve excitation propagating along a nerve fiber it would be possible to get deep inside the nature of this process.
* Mitogenetic radiation – a weak ultraviolet emission accompanying different biochemical processes. |
I cannot say that the work in Gurvich's laboratory was quite successful for me in spite of his kind attention to my friends and me.
The thing is that the method employed in his laboratory for registration of the mitogenetic emission was based on stimulation of cell division in yeasts. So each experiment required estimation of the number of budding yeast cells and comparison of this parameter in normal cells. This was a rather subjective and labor-consuming procedure. In some cases such calculation was successful, whereas in other cases it was not. Ms. Gurvich specially taught us, but the results of estimation were nevertheless very unreliable. In spite of enormous efforts and time spent, there was little progress in that work.
As to me, I was not sure that my results would be reproducible and that they did represent «objective reality». I was not sure when the experiment was quite reliable and when not. This stimulated my search for methodological tools that would give me confidence in reliability of both methods used and results obtained in my experiments.
I think that this search together with some other reasons (which I did not realize at that time or did not want to realize at the subconscious level) turned me toward biochemistry. Biochemistry, the so-called test-tube biology, employing a chemical approach for analysis of biological phenomena, looked more reliable to me. It provided a firm methodological background required for my sense of satisfaction. This was the major factor that influenced my decision to change the area of my scientific interests.
I should also say that in the second half of the 1940s it became clear that the nature of biological processes would be understood on the chemical level rather than on the energetic or physical levels. Intention to understand the nature of biological processes (including nerve processes) would definitely be directed toward investigation of chemistry of these processes. At that time I did not understand this point consciously, but I definitely felt it subconsciously.
I also had some problems with experimental work employing such biological objects as a neuromuscular preparation or experimental animals. Possibly such work requires a «special personal arrangement» of a scientist. I think that a good physiologist should be a skillful surgeon strong enough to perform vivisections. My personality did not meet such criterion. I was more interested in the field of the «test-tube biology», elemental chemical processes, and in chemical rather than surgical or physiological methods. And, of course, I wanted to get reliable and reproducible results from my experimentation. Thus, in the third year of my university studentship I desperately wanted to specialize in biochemistry. So, I made an application to the Chair of Animal Biochemistry and would have definitely continued my education at this chair, but suddenly (I cannot explain why!) the Chair of Plant Biochemistry attracted my attention. The reason was that my deeper study of biochemistry resulted in understanding of the fact that I became more and more interested in this science as such rather than a useful tool for investigation of nerve processes. Biochemistry as the basis for understanding the nature of life became my major interest. In this respect plants were much more interesting objects than animals, because plant biochemistry was much more «diverse», and it started from the very beginning of life, photosynthesis, the synthesis of organic matter. Elemental biochemical processes in plants are also much more diverse and variable than in animals. (At that time biochemistry of microorganisms was also referred to as plant biochemistry.) I was deeply interested in these processes.
There was another reason by which I chose the Chair of Plant Biochemistry. I mean styles of learning biochemistry at the Chair of Animal Biochemistry and the Chair of Plant Biochemistry. In animal biochemistry a «ladies» style dominated. Its chairman, the famous scientist Professor Sergey Severin, was a brilliant lecturer, and we all attended his university course of lectures in biochemistry. However, his associates and various assistants involved in the education at this chair almost deified him. They taught their students very meticulously and scrupulously. They accepted a word from the professor as unquestioned dogma and his viewpoint as «the truth of the final instance».
At the Chair of Plant Biochemistry, directed by Academician Alexander Oparin, there was a completely different style because of the relatively young Professor Andrey Belozersky, who was in charge of the educational and pedagogical processes.
Soon after the beginning of my specialization in animal biochemistry, I realized that I desperately wanted to continue my education at the Chair of Plant Biochemistry. So I made an application to this chair to accept me for subsequent specialization. It was a rather difficult procedure, which was not well understood by my friends, teachers (and to be honest, by myself). However, I felt that I needed to change chair while it was not too late.
In the very beginning Professor Belozersky was rather skeptical of my intention to change chair after the beginning of an academic year. He tried to persuade me to leave the situation just as it was. He explained that research is not as romantic as I had suggested and hard-working ability was the major factor, while philosophy and biophysics were good of course. Real research requires solid reproducible observations. The most important in our work is the «coincidence of duplicate determinations», he said.
Professor Belozersky was a wonderful person and influence on my scientific fate and my scientific development. I feel that my scientific viewpoints, style of work and human relations, criteria of evaluation of results, reference group on which I oriented and began to orient later, as well as personal self-evaluation were initially formed under the strong influence of Belozersky's personality. (I am not sure that he realized such influence of his own personality.)
Professor A. N. Belozersky (center) and docent N. I. Proscuryakov (right) with students of plant biochemistry chair. |
Belozersky was a very talented, charming, and benevolent person. He was a real scientist. He was real in everything. Being a genuine researcher, he belonged to a cohort of investigators who «extracted» primary facts hidden in nature. He had surprisingly simple viewpoints on many problems, common sense in evaluation of scientific events, results, and people. He demonstrated extraordinary respect to students, colleagues, and other scientists. Belozersky was characterized by such a wonderful feature as «presumption» of respect to everyone and everything. He was very delicate. His intention not to offend persons (especially students), complete lack of haughtiness and snobbism, and simultaneously his accessibility but without familiarity were very typical of him. These characteristics of Belozersky were wonderful and absolutely natural. In the very beginning of my studentship at the Chair of Plant Biochemistry I was not very successful, especially in learning the «art» of experimental work. (At that time it was definitely art rather than ordinary skill.) Experimental work was very empirical and strongly depended on «good hands», scientific «flair», and the sixth sense with respect to the material used. I found perfect understanding of my problems, some indulgence, healthy humor, i.e. normal human relations. I think that Belozersky with his large experience well understood how touchy a young student is under these circumstances. So all that I accepted from Belozersky was «grown in a well prepared and very sensitive background». He never taught us «do this or that». He never taught what is right or wrong. He just commented on our activities, discussed our problems with us, gave his advice and recommendations, and that was all. All features of his personality influenced me (and others as well): his direct evaluations of my work, his comments, lectures, talks, short remarks during conversations, etc.
One of the main understandings which appeared there and which basically predetermined my scientific carrier was the sense that I should work as hard as I can and as much as possible. I also began to realize that real experimentation requires knowledge of a research object, possession of the art of experimental work, reliability, and reproducibility of results obtained and methods employed. Evaluation of results obtained by yourself and your colleagues also required some proportion of healthy significance and simplicity.
I think that I came to such understanding mainly due to communication with Belozersky, and I am confident that this understanding is still with me Professor Belozersky treated me kindly, maybe too kindly, but it was typical of him: there were just a few male students at his chair. Such kind attention to his male students was maintained during the whole studentship and even afterwards up to the end of his life. He always helped me and was involved in my fate and my work. He was always benevolent, ready to help and advise.
Later he involved me in work at the university, where I still teach students at the Chair of Virology, which he founded. I am very grateful to him for his kind attention to me.
My diploma project was about nuclear proteins. I thought it would be a very interesting project, and indeed it was. It was the first continuation of initial observation by American researchers, Mirsky and Pollister, that nuclei of plant cells as well as nuclei of animal cells contained histone, the basic protein of chromosome structure. At that time this fact was not known; moreover, it was believed that the presence of histone was restricted to animal cells. However, using some remarks made by Mirsky and Pollister and employing their method, I demonstrated that plants also contained histone. In my diploma project I isolated large amounts (900 mg!) of this protein, analyzed its amino acid composition, and thus provided convincing evidence for the existence of histone in plants.
Belozersky was deeply interested in this work. However, it was performed in 1949–1950, right after events accompanying the sadly known session of the Academy of Agricultural Sciences headed by Lyssenko. These events significantly disturbed development of research related to nucleic acids, chromosomes, and nuclear proteins in the USSR. This was a time when real normal scientists, classical biochemists, tried to avoid research projects related to such areas. Belozersky was not an exception: he had many problems because of his works related to nuclear proteins, DNA, and his contacts with classical genetics at the university. So he did not want my project to be done at his chair, and he suggested projects on bacterial antigens. However, I had no choice and finally we succeeded to obtain approval for the project thanks to A. I. Oparin. He had just obtained a large project for the whole chair on oak as the basic tree for the so-called field-protecting forest strips; this problem was quite popular and «fashionable». (Such field-protecting forest strips dominated in the «Stalin plan for reorganization of nature».) Oparin suggested that the nucleoprotein problem could be investigated using oak as the research object. Of course, it was difficult and inconvenient to employ oak as the research object, but the major thing was that the nucleoprotein project could be started.
I began to work on this project (with oak as the research object), but several months later I continued my work using real research material – wheat germ – and obtained interesting results on histones. I was totally inside the problem of nuclear proteins and their role in cell differentiation and mitosis. Although Professor Belozersky worked for the N. F. Gamaleya Microbiology Institute* as a consultant and worked on bacterial antigens, I had no interest in immunology. Diploma projects of my friends dealt with bacterial antigens, but those problems were absolutely out of my interest. Moreover, I did not accept Belozersky's proposal to work on bacterial antigens because I was keenly interested in nuclear proteins, nucleoproteins, and global problems of cell division and differentiation. I was confident that this was real biochemistry.
* Institute of Epidemiology and Microbiology named after famous microbiologist N. F. Gamaleya and belonging to the USSR Academy of Medical Sciences. |
I graduated from Moscow State University in 1950. It was a difficult time and I had to join Professor I. B. Zbarsky, who was Head of the Biochemical Laboratory at P. A. Gertsen Research Institute of Oncology. He was interested in tumor cell nucleoproteins, particularly in the so-called tumoroproteins, putative tumor-specific proteins. (A hypothesis on tumor-specific proteins and their role in tumor cells was proposed by his father, B. I. Zbarsky, an outstanding Soviet biochemist, Chairman of Biochemistry at the First Moscow Medical Institute and also Director of the Lenin Mausoleum Laboratory.)
My relations with I. B. Zbarsky had a good start: being a student, I gave a talk at his laboratory seminar, and he wanted to see me in his laboratory. I also wanted to continue my work on nuclear proteins even if from different object(s). For me it was important to continue research work within the same problem irrespectively to research objects.
To exclude all possible complications during my postgraduate employment, Dr. I. B. Zbarsky asked his father to make an application to the University Postgraduate Employment Commission to send me to the Lenin Mausoleum Laboratory, which was organized to look after Lenin's body for prevention of any signs of its decay. (Here I should explain that due to free of charge education in the USSR, the graduates had to work for at least three years for organizations that sent their applications to the University Postgraduate Employment Commission. This commission «distributed» the graduates between applicants.) I. B. Zbarsky promised that in spite of my position at the Mausoleum Laboratory, I would be working at the Institute for Oncology. I accepted his offer because I relied on his experience. So such application was made, but it played a bad role in my fate. Entering the office of the University Graduate Employment
Commission, I felt that things had gone a way I did not want. Members of the commission explained that they were not satisfied with the application from the Mausoleum Laboratory because employment at this laboratory requires special selection. I was told that I could be sent to any other laboratory. Here I should say that an anti-Semitic tendency was just beginning in science in contrast to art, literature, or theater, but together with my low social (political) activity it was sufficient reason to prevent my entrance into the Mausoleum Laboratory. I thought that all my «rosy» perspectives collapsed in one moment, but Belozersky immediately began to investigate possibilities of my postgraduate employment. He quickly found that Dr. V. A. Blagoveschensky, a biochemist and bacteriologist who was organizing the large Biochemical Department at the Gamaleya Microbiology Institute, needed junior staff members. They rapidly made the appropriate application offering me a position of junior technician. Dr. Blagoveschensky treated me very kindly. As I was a student of Professor Belozersky, he permitted me to continue my university project on nucleoproteins, but on bacterial objects.
In the Laboratory: Searches and Findings
Thus, in summer of 1950 I began my work at the department of Biochemistry, Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences of the USSR.
Dr. Blagoveschensky was interested in problems of vaccination and bacterial antigens, but I was indifferent to them. On the third floor of that institute there was a department directed by Professor L. A. Zilber, an outstanding scientist who was successfully working on cancer immunology. He was basically one of the founders of this discipline.
Although I had no interest in immunology there was one important point that attracted my attention. Zilber not only developed immunology of cancer, but actively investigated nucleoproteins and nuclear proteins of tumor cells. Based on his theoretical considerations originating from the viral theory of cancer, he believed that specific tumor antigens ought to be found mainly among nucleoproteins. (This is because viral proteins should be associated with the nucleoprotein fraction.) This explains why biochemists of his laboratory were working on tumor nucleoproteins. A. N. Belozersky was a consultant in these studies carried out by Z. A. Avenirova and V. A. Artamonova.
In that year the University Graduate Employment Commission sent four graduates to the Gamaleya Institute. Dr. Blagoveschensky could not accept all of them. I still remember the episode when we, four young specialists, were in the office of the Staff Department. The head of this department phoned to Zilber and asked him to come to his office and to choose some of us for work in his laboratory. Zilber came. He was a big, tall, confident, and very impressive man (a man from another world!). He briefly interviewed each of us, asking about our diploma projects and our supervisors. He was going to accept me, because I was a pupil of Belozersky, who was a consultant of some projects in his laboratory and whom Zilber highly evaluated and respected. He also appreciated that I had good background in the field of nucleo-protein research, and his laboratory used the techniques developed by Belozersky.
However, Blagoveschensky refused to send me to Zilber's laboratory. They had a hot conversation, and Zilber was offended by his decision and refused to accept anybody.
Thus, starting my work with Blagoveschensky, I already wanted to work with Zilber because nucleoprotein research had top priority in his laboratory. It was a reasonable intention of a young researcher to join a laboratory in which scientific problems of his interest represented «the main stream» of its research as well. In Blagoveschensky's laboratory the nucleoprotein project was given to me just due to good relations between Blagoveschensky and Belozersky.
Anyway, I started my work in Blagoveschensky's laboratory. However, one year later Blagoveschensky was invited to work in another bacteriology institute out of Moscow, and he left the Gamaleya Institute. The next head of that Department, Professor V. S. Gostev, was a completely different person. To begin with, he was a disciple of Zhukov-Verezhnikov, a powerful microbiologist supporting Lyssenko's views. He was interested in completely different studies, whereas I desperately wanted to continue my work in Zilber' department.
In Zilber's Laboratory
Immunological methods developed by Zilber for detection of specific tumor antigens (anaphylaxis with desensitization) looked very attractive to me because it opened new horizons in studies of specificity of nucleoproteins, their functioning, and isolation of tumor-specific nuclear proteins. All these studies were carried out in Zilber's laboratory. The other reason was Zilber's personality.
He was an outstanding scientist involved in solution of very interesting problems. It was clear for me that my work with him would find resonance and support and would attract interest to my research, which I was limited in at that time. It is very natural for a young researcher to have a very competent, very distinguished supervisor who would be interested in your work, who would encourage you to overcome difficulties and problems in your research. Simple benevolence is good but it is not enough.
These were my reasons, which drove me to move to Zilber's laboratory or at least to have a joint project with him. I established contacts with his biochemists, and we discussed possibilities of a joint research project. This was right after
Blagoveschensky left and Gostev appeared. Gostev sharply objected to any contacts with Zilber. He was also interested in tumor immunology but had «cold relations» with Zilber. I do not think that this was due to competition in science, because Zilber's personality was much higher in all respects. Gostev's ideals were very far from the ideals of my university teachers, Belozersky, or Gurvich. He was a completely different person.
Although it was clear for me that I would do my best to join Zilber and his team, I discussed this problem with my friends, my wife, and of course with Belozersky. He was the only person who strongly recommended me to go to Zilber. He said it would not be easy to work with Zilber; many people believed that he was very autocratic and even a despotic person, but I would be happy to work with him. Belozersky said that Zilber was a very enthusiastic man actively working in science and we would easily establish our relations. This was the only definitive viewpoint. Most of my friends were rather skeptical of my intention to change laboratory. They argued that I was a professional biochemist, working just at the Biochemical Department. Moving to Zilber I would be surrounded by specialists in medicine, immunologists (basically non-biochemists). They felt that I would do all biochemical studies by myself, and nobody would help me in solving biochemical problems. But I ignored these skeptical warnings.
Thus, I directly applied to Zilber asking him to accept me as a member of his department. Zilber was keenly interested to accept a student of Belozersky into his department, but it was very difficult. There were long-term negotiations (for several months) with the institute director, Professor V. D. Timakov, but finally this lucky event happened, and I became a member of Zilber's department. This change in laboratory was also accompanied by a better position: I became a senior technician with a higher salary. (This was also important because I had family.) However, the final result was that I had come to a laboratory that needed my work and was keenly interested in it.
Thus, in the very beginning of 1952 I began my work in Zilber's department. This was a very important and decisive event in my life. I started my work from fractionation of tumor nucleoproteins. Fractionation was very important for isolation of tumor-specific antigens under control of immunological tests developed in that laboratory. In the very beginning of my work I was not familiar with these tests, and so I worked in close contacts with medical immunologists Ms. Zinaida Baidakova and Dr. Nikolay Narcissov. (They were the staff members closest to Professor Zilber.) The anaphylactic reaction with desensitization (A-D) was the major test for tumor antigens. Guinea pigs were initially sensitized with tumor nucleoprotein; three weeks after sensitization the guinea pigs were desensitized with gradually increasing doses of nucleoproteins isolated from normal tissues. When the guinea pigs were totally desensitized to normal antigens, they received a single dose of initial tumor nucleoprotein. Usually such administrations were accompanied by anaphylactic shocks of various intensities. This suggested the presence of some tumor antigens specific for a particular tumor and absent from normal tissues. This rather simple but elegant and convincing method developed by Zilber and his team brought major success in that direction of tumor immunology. This method represented the important tool employed in Zilber's research. So I fractionated tumor nucleoproteins on the basis of this reaction. I was especially interested in nucleoprotein fractionation based on differential centrifugation. Several years earlier such approach was proposed by the Belgian biologist Albert Claude. Employing this method it was possible to isolate ribonucleoprotein-enriched cytoplasmic granules, nuclei, and non-structural cytoplasm. For this purpose G. S. Bezverkhyi, an engineer who used to work with Blagoveschensky, and I adapted milk separators for isolation of cytoplasmic granules (mitochondria and microsomes), and then we applied our method to nucleoprotein fractionation.
Pilot results were clear and promising. They suggested special activity and the presence of specific antigen in the cytoplasmic granule fraction. However, subsequent results were less stable and reliable; their reproduction depended on anaphylactoid reactions (i.e. false anaphylactic reactions induced by protein aggregates or cell debris), which were obtained using these corpuscular, poorly soluble fractions. I was upset and depressed about not being able to reproduce some results and the conclusions, or lack thereof, we had to draw from that fact.
I worked very hard and tried to use different variants of the method. I also started parallel work with Dr. Narcissov. Using complement-fixation reaction with sera of tumor-bearing rats, the results became more stable, reliable, and reproducible, and the reaction itself was much simpler. So, under control of complement-fixation reaction Narcissov and I provided convincing evidence for the localization in the mitochondrial/microsomal fraction of an antigen responsible for reaction with sera obtained from tumor-bearing rats.
That was the time (1952) when immunogenetics was formed as a science. It became clear that such studies would employ a so-called syngenic system (i.e. a system of genetic identity of tumor donor and recipient, although this term appeared later.) We started to think about various controls and tried to work on primary tumors and sera of the same animals. Generally we confirmed the major conclusion that during growth of sarcomas induced by carcinogens (primary or transplanted sarcomas), the sera obtained from the tumor-bearing animals exhibit complement binding reaction with extracts from the same tumors, mainly with the cytoplasmic granule fraction isolated from these tumors [1].
In parallel work, together with a group of engineers from the Research Institute of Chemical Machinery, we tried to improve the separators. This work culminated in several models of reasonably good separators applicable for isolation of cytoplasmic granules. Furthermore, we developed a new type of separator, the chamber separator, which was able to separate particles from the flow of tissue homogenate by their size and density. Thus, we were able to use both differential centrifugation and density gradient separation in one procedure. (I should say that using our device it was possible to work with large volumes of starting material.) This was reasonably good work of 1955. The problem was that there were just one or maximally two copies of our separators, and our work could not be further developed [2]
Thus, in the 1950s I was involved in all studies carried out by Zilber's department. I worked on separators, fractionation, and analysis of antigenic properties of various intracellular components, and also I learned cancer virology and immunology. In parallel, I set up the method of free electrophoresis after Tiselius and employed it for characterization of proteins obtained during fractionation of cell components. Together with Narcissov, we studied antigenic properties of these fractions using sera obtained from tumor-bearing rats.
In 1955 I received the Ph.D. degree. However, I should say that the title of my thesis, «Study of Some Fractions from Tumor Tissue», was a bit boring.
At that time I was interested in immunology mainly as a tool for studies of cell structures, tissue-specific proteins, and a tool for recognition of proteins specific for tumor tissue. Immunology attracted me as a method that would help solve biological and oncologic problems related to cell differentiation. I was poorly familiar with immunology and spent much time in attempts to understand it. There were a few handbooks of immunology, but they had a strong emphasis on medicine and microbiology. So it was difficult for me to understand the main notions and principles of immunology. (Literally speaking, all immunology of that period «sank» in abundant complex medical terminology, which often substituted real understanding of actual scientific links by numerous unrelated phenomena.) Zilber's seminar organized in the Gamaleya Institute for medical aspirants (which was also attended by senior technicians) was very helpful for understanding immunology.
Basically, Zilber clarified many problems of immunology for me. I still remember that I started to understand the unitary theory of immunity: the reaction between antigen and antibody represents the basis for all multiplicity of various phenomena joined by immunology, and secondary immunological manifestations can have various phenomenological features.
The Russian translation of the book by William Boyd «Principles of Immunity» (or possibly «Principles of Immunology») published in 1947 (if I remember correctly) also helped me in learning immunology. This very good textbook with emphasis on immunochemistry clearly described the principles of immunity.
Only reading this book and participating in Zilber's seminars cleared my brain in terms of immunology, and I began to understand the basic principles of this science. In 1956 William Boyd visited Moscow, and he had many discussions with us, members of Zilber's department. Looking after him in Moscow, I asked him how he managed to write such a wonderful book, describing basic principles of immunity in a simpler way compared with other textbooks. He said that it was difficult for him (a chemist interested in immunology) to get inside the immunologic literature, very complicated with phenomenology and immunological notions and terms. So he started to learn principles of immunity from the chemical viewpoint and later, when he became well familiar with them, he wrote that book. Perhaps a reader lacking medical and immunological background needed such logics of a «xenobiotic» expert for understanding this science.
The second edition of this textbook was also translated into Russian. However, it was larger and more complex and was not as clear as the first edition both in construction and style of presentation.
Immunodiffusion
In the middle of the 1950s I became familiar with the method of antigen precipitation in gel. In laboratory seminar we discussed the method of Ouchterlony applicable for analysis of tissue proteins. In two papers by B. and V. Björklund published in 1952, this method was used for studies of some tissue proteins. We actively discussed applicability of this method for detection of specific tumor antigens. We understood the great possibility and facilities of this method, its resolution and analytical power. We started to realize that this method might be used for studies of individual antigens, individual proteins (without their purification from a complex mixture), for comparison of these proteins, for elucidation of not only species but also tissue (including tumor) specificity.
Nevertheless, working with anaphylaxis we believed that the method of gel precipitation was rather crude and insensitive for detection of fine differences, which do exist between the antigenic structure of normal and tumor cells. This was our strong belief. However, I suggested that this method was very valuable for us anyway, because it could be used to compare antigenic structure of different cytoplasmic structures and to identify proteins characterizing nuclei, cytoplasm, and various tissues. I was confident that this method opened «colossal» facilities for identification and characterization of complex protein systems. There was very attractive promise for use of this approach as such (or at least as an intermediate stage) for recognition and identification of specific tumor antigens that were the basis of our studies.
However, until 1955 I was working hard on the above-mentioned projects and analysis of the data obtained and preparation of the Ph.D. thesis. Afterwards I continued my work on the chamber separator and electrophoresis and analysis of sera from tumor-bearing animals. I did not have time to use the gel precipitation method in my research.
In 1956 Dr. Vera Parnes, a senior researcher of Zilber's laboratory (who was also interested in the gel precipitation reaction), and I decided to set this method together. I had to work on the immunochemical part of our common project, manipulations with gels, fractions, antigen preparations. Her part of that work included selection of corresponding antiserum, potent precipitating sera, and system of normal and tumor tissues that we would compare.
A very important thing was that we had an excellent precipitating serum, obtained by Z. L. Baidakova for a completely different purpose: for preparation of an antileukemic vaccine. She obtained very potent donkey antiserum against human leukemic spleen, which we also used with Parnes in our manipulations during reproduction of the gel precipitation method.
Finally, we were able to distinguish the first precipitation band in gel and first reaction of identity. Everything was new for us: agar preparation (starting from alga), preparation of agar wells, and protection of this system against bacterial growth. In other words, everything was difficult, but the major result was that this method worked and its use yielded clear bands. This was perfectly wonderful!
Besides the experimentations with leucosis, Avenirova and I began to compare the antigenic structure of normal liver and hepatoma. Using separators, we isolated several subcellular fractions (mitochondria, microsomes, nuclei) and compared them with each other and also with normal liver and hepatoma. We obtained very good precipitation bands with antisera against liver mitochondria and microsomes, against homogenates of normal and tumor hepatic tissues. In 1957 we had two systems in which the precipitation in gel gave results: the leukemic system used in studies with Parnes and the hepatic system employed for joint studies with Avenirova. Parnes continued the work with leukemia spleen, while we together with Avenirova focused our attention on the hepatic system.
The hepatic project ran rather well and a question arose: is this method applicable for detection of differences between normal and tumor tissue, or is such precipitation too crude for detection 19 of such fine differences? In other words, is this wonderful method applicable for detection of tumor tissue specificity? This issue be came the major goal of our studies.
I was confident that even if the sensitivity of this method would be insufficient, it might give us purer and better characterized tumor tissue fractions that would be further used in the anaphylactic reaction.
We began to compare cytoplasmic granules from normal hepatic tissues and hepatoma. All this work was carried out on mice. Hepatoma employed in our experiments was obtained by V. I. Gelstein. She spent some time working with Zilber and was well familiar with his studies. We had very good contacts. We started comparing the antigenic structure of normal and tumor hepatic tissues and obtained promising results almost immediately. During direct comparison of mitochondria and microsomes from normal liver and hepatoma, we found lack of at least one or even a few antigens present in the normal tissue.
In 1956 E. Weiler, from West Germany, described the phenomenon of «antigenic simplification». Comparing antigens of normal liver and hepatoma using the complement binding reaction, he demonstrated loss of the tissue-specific liver antigens in hepatomas. Weiler defined this phenomenon as «antigenic simplification». This antigenic simplification was immediately detected in our experiments with precipitation in gel. We found that several tissue-specific antigens typical for normal hepatic tissue disappeared in hepatoma. This phenomenon was potent, clear, readily reproducible, reliable, and very promising. It was possible to isolate these antigens under control of reliable test and investigate their behavior during chemical carcinogenesis in various tumors. Basically this opened wide prospects for studies of this phenomenon.
We were very lucky, and in collaboration with Vladimir Tsvetkov we developed a simple method for purification of one of the tissue-specific hepatic antigens, which we name AO-antigen, the antigen of organ specificity. This was a rather simple and good method based on ultracentrifugation and electrophoresis, which we set up in Zilber's laboratory. This method yielded immunologically pure antigen with high activity and very low-protein content. Isolation of an individual protein was a very difficult task, but we had such an antigen. We were able to investigate semi-quantitatively the fate of this antigen in various tumors during chemical carcinogenesis. This antigen demonstrated a direction for subsequent studies employing other tissue-specific antigens (see Ref. [3], pp. 308–313).
This work was quite nice even by modern criteria, but at that time when individual antigens were not available, its continuation seemed to show promise. I thought that we might spend time for working on this project, but Zilber had another viewpoint. He often said to us that it is good interesting work, which definitely opens a new chapter in immunology, but specific antigens are much more important. He «pushed» us toward tumor-specific antigens, which could be recognized using the same technique. He used to say that we could continue our research on the organ antigens at another time.
The work on specific antigens was stimulated by another point, which was also rather important for Zilber. These events took place in 1957 and in the next year he would have to present results of his department at the 7th World Cancer Congress in London. This was his first international presentation after a 30 year forced interval, and he definitely wanted to demonstrate tumor-specific antigens to the international audience. So Zilber stimulated us to work on these specific antigens. This was a much more difficult project compared with our work on the organ-specific antigens, because such tumor-specific antigens were hard to detect. However, there were some hints in our experiments: one band (which we considered as the band of specific antigen) appeared in some experiments and disappeared in others. So we postponed this work for experiments with more reliable antigens. However, Zilber insisted, and finally we started our «pursuit» of this antigen «weakly twinkling» in the spectrum of antigens common for normal and tumor tissues.
One of the major difficulties in the detection of specific antigens was the fact that specific differences could be detected on gel plates within a peripheral zone of antibody–antigen interaction, where this reaction is minimal, looking like a weak spur. So I was thinking of an experimental protocol in which specificity could be detected in the zone of optimal interaction between antigen and antibody, where the precipitation zone would be most pronounced. Finally we succeeded in developing a system now known in the literature as the «quadrant scheme». Using this scheme it was possible to (1) compare two complete antigen–antibody systems and (2) displace specific reaction to the most potent reaction zone [4]. This approach was very successful; it is still employed now, and since 1957 commercial firms issuing matrices for precipitation in gel include the quadrant scheme into their kits. I was very pleased that this work was appreciated and gave an expected result. We obtained clearer detection of the hepatoma specific antigen and reliably detected its line in the complex spectrum of bands. In the summer of 1958 at the World Cancer Congress in London, Zilber presented the results of studies on tumor and normal antigens obtained by means of precipitation in gel. He demonstrated the phenomenon of antigenic simplification, and in the spectrum of precipitation bands he also showed the band corresponding to specific hepatoma antigen [5]. We continued our successful studies and a very good period in our research appeared. As I wrote above, we developed a method for purification of one of the most potent organ-specific liver antigens (AO) and demonstrated association of this antigen to particulate fraction and its easy solubilization. Now I am confident that properties of this antigen and the method for its purification well fit cytokeratins.
Here I should say that the phenomenon of antigenic simplification was not as universal and abundant as Weiler believed in the very beginning, and as we suggested in the period of the first experiments.
Our subsequent experiments on the behavior of the organ-specific antigens in primary tumors and at various stages of carcinogenesis revealed that tumors had certain features of their organ origin and contained organ-specific antigens [6].
However, hepatoma-22a was the most non-differentiated marginal form and so all changes were highly expressed. (We were quite lucky to use this research object.) So it was reasonable to continue this work using various tumors.
Individual Antigens and Monospecific Antibodies
Our major achievements of that period (besides organ-specific antigen purification and the quadrant scheme) consisted of two more important steps in characterization of our antigens. These were preparation of monospecific antibodies to individual organ and tumor-specific antigens and the development of the method of immunofiltration for immunochemical purification of these antigens.
We obtained monospecific antibodies by dissociation of specific precipitates. Specific precipitate containing one antigen and corresponding antibody was obtained using a multicomponent antigen system and polyspecific antiserum. Using successive absorption of this serum by unrelated antigens and partial purification of the investigated antigen, we finally obtained the precipitate formed by just one antigen–antibody pair. Subsequent incubation of this precipitate in weakly acidic medium resulted in elution of 15% of the antibodies. This procedure was rather complex and time-consuming, whereas antibody yield was low. Such system exhibited different behavior with different antigens, but to our surprise (and satisfaction!) we obtained a whole spectrum of antibodies against various antigens. This work was carried out in collaboration with N. I. Khramkova (Kuprina).
The initial work on eluates was carried out with Z. A. Avenirova (see Ref. [3], pp. 308–323). We easily obtained monospecific antibodies to AO and to our hepatoma-specific antigen. This was perfectly wonderful because using such antibodies it would be possible to detect an antigen of interest in the whole tumor extract or in extract of tumor subcellular fractions: precipitation in gel gave clear and well-detected band of the antigen, which could not be missed among other bands.
Characteristics of the eluate of antibodies to the specific hepatoma antigen. EH, eluate of antibodies to hepatoma antigen; AH, anti-hepatoma serum; eN, eluate to the organ-specific liver antigen, AO; gH, gamma-globulin, isolated from mono-specific anti-hepatoma serum; AN, anti-liver serum; AS, anti-splen serum; AK, anti-kidney serum; H, hepatoma extract; N, liver extract; S, spleen extract; K, kidney extract. From Ref. [3]. |
This provided a firm and reliable background for our subsequent studies. Using such a reliable test, we continued our work. In 1957 N. V. Engelhardt joined our group after graduating from Moscow State University. At that time V. S. Tsvetkov also joined our group, and we developed the method of organ-specific antigen purification with him (see above). A. I. Gusev worked in close contact with our group. Using the same methodology and objectives, he started to work with Rous chicken sarcoma as the model. Gusev was «a master» of experimental work, and he also obtained very clear results with gel precipitation and antibody eluates. We had very good and very open interaction, and in 1958 our group of five researchers continued very intensive studies on tumor antigens.
Together with Tsvetkov, we started to develop the method of immunofiltration. We considered the following hypothesis: our antigen (which we called H-antigen, antigen of hepatoma) was characterized by rather high-electrophoretic mobility, similar to that of serum alpha-globulin, i.e., larger than that of gamma globulin (to which antibodies belonged). So using electrophoresis in agar it was tempting to create electroosmotic movements of antibodies to meet antigen, which would move in opposite directions: antibodies to cathode and antigen to anode. In this case the antigen had to pass through the antibodies. If the antigen contained «irremovable» electrophoretically similar contaminants common for normal and tumor tissues and gamma-globulin fraction contained antibodies against common components of normal and tumor tissue, such antigen–antibody movement in opposite directions would represent antigen «filtration» through antibodies. In this case all contaminants would have to bind to corresponding antibodies and precipitate or form antigen–antibody complex characterized by lower electrophoretic mobility than the antigen. Consequently, only pure antigen was able to penetrate through the «antibody filter», whereas all contaminations would be retained on it.
Initially we developed an analytical version of such system, then we made a preparative version, and finally within one year (or even a few months) we developed several variants of the immunofiltration method. Using this method we obtained immunologically pure antigen of hepatoma. It exhibited one precipitation band with polyspecific serum against hepatoma and did not react with antiserum against liver, and it was not detected in other organs. Thus, it met all our criteria [7].
Using this method applicable for most various antigens, we obtained specific hepatoma antigen.
Immunoelectrophoresis of specific hepatoma antigen, isolated by immunofiltration. From Ref. [3] |
We were very proud to have in hand the first pure tumor-specific antigen.
However, we faced other problems: what is the nature of our antigen, its origin, and putative role (if any) in antitumor immunity?
So far we had been solving methodological problems: would precipitation in gel be applicable for detection of specific antigens, would it be possible to develop methods for isolation and identification of this antigen? When antigen isolation was the major goal of our studies, we did not think about the nature of the antigen. We thought that when this antigen would be available our experimental system would give us a hint. However, in the process of antigen isolation we became more and more concerned about its nature and putative role. First, this antigen might be an antigen of a «passenger» virus, which simply «settled» in the tumor. In such case it is natural that this antigen is detected in tumor but not in normal tissues. This might be the case especially if we took into consideration that this antigen was found mainly in our transplantable hepatoma and rather rarely in primary hepatomas induced by carcinogen. This increased the possibility that this antigen would be a secondary antigen indirectly linked to tumor transformation.
Furthermore, we were not confident that our hepatoma actually represented hepatocellular cancer (i.e. the tumor originating from liver parenchymal cells representing the major population of liver cells). It might be possible that the tumor originated from bile duct cells, which are much less abundant in liver. If our antigen represented the organ-specific antigen of bile epithelium, we would not detect it in the liver due to low sensitivity of the gel precipitation method, and in tumor its content would be much higher. In such case this antigen would be just bile epithelium organ-specific antigen rather than specific tumor antigen, which we were looking for. (This possibility was quite real and our colleague Dr. A. Ya. Fridenstein suggested it as a plausible alternative explanation of our findings.)
Finally, it was also possible that small amounts of this antigen present in normal liver were below the detection limits of our method, whereas in the tumor the antigen content was much higher.
In the end of 1960 we started analysis of all these possibilities. We were very skeptical and expected that one of these versions would be experimentally confirmed. We developed a highly sensitive gel precipitation method, which were also based on immunofiltration. This method was 10–20 times more sensitive and could readily detect possible traces of this antigen in normal liver.
N. V. Engelhardt started to adapt an immunofluorescence method for investigation of localization of our antigen. This was a very difficult methodological task, but we cherished hopes for use of the immunomorphological method, which would detect possible localization of our antigen in bile capillaries. However, this very difficult task was solved only in 1969 (see below).
Simultaneously, we started to analyze various tumor strains for the presence of our antigen. This would give us a clear answer whether this antigen was linked to some «passenger» virus, which might be recognized in other tumors.
These were our major goals of experiments of 1960–1961, which gave us variable results. At that moment one event sharply changed direction of our studies.
Alpha-fetoprotein (AFP)
In parallel with investigation of hepatoma-specific antigen, we also continued our work on «antigenic simplification». Our results indicated that antigenic simplification was more complex than suggested by Weiler and by us (studying hepatoma-22a only). In other hepatoma strains organ-specific antigens persisted, and later we also detected traces of one organ-specific antigen in our 22a hepatoma.
Tumor progression is characterized by changes in the pattern of organ-specific antigens; each tumor has its own set of organspecific antigens. We identified at least seven various antigens. This was the «zone of responsibility» for Mrs. N. I. Khramkova (Kuprina).
We observed very interesting phenomenon of individual anti-genic structure of various hepatomas (see Ref. [6]). We decided to investigate this individuality in more details, whether it reflected the possible existence of discrete periods in the development of liver characterized by certain sets of organ-specific antigens. It might be possible that antigenic «maturation» of the liver has several discrete stages, and structures of different hepatomas might correspond to different stages of liver differentiation. We had a collection of antibodies against seven organ-specific antigens (i.e. test systems for these antigens), and Khramkova and I decided to check this hypothesis. Since we also had a test system to the hepatoma antigen, we also employed that system as well. We had no hopes – we just wanted to try.
In our laboratory there was a young student from the medical institute. She wanted to become familiar with immunology and to get some experience in laboratory work. She learned to perform precipitation in gel, and Khramkova gave her all our test systems. The student used embryonic liver for analysis of organ-specific antigens. We had never analyzed them in embryonic liver. She collected embryos at various stages of development and performed experiments with all the test systems.
However, that student disappeared from our laboratory for some unknown reason, and we had to analyze her precipitation results ourselves. We were shocked! In the extract from the embryonic livers there were huge amounts of our «hepatoma antigen», which we never saw in the hepatoma preparations. Our first feeling was that this was experimental error of our student. However, we did not have any plausible explanation for mistakes she could make! We immediately repeated that experiment and it demonstrated exceptionally high content of our antigen again. We repeated this experiment using the liver and other embryonic organs and results clearly demonstrated very high levels of this antigen in them! These levels were much higher than in hepatoma extract!
At first glance this was a dead-end. We did not have any plausible ideas on the origin of this antigen in the embryonic liver. Why were its significant amounts detected in other embryonic organs? Suddenly we realized that in reality this liver antigen might be blood antigen (serum antigen). This would explain its presence in various organs because we did not wash these organs free from blood! We immediately investigated embryonic blood serum and obtained very high titer of this antigen; we never detected such high titer in any other biological material used for analysis!
It became clear that the former hepatoma antigen, (in reality the so-called hepatoma antigen) represents blood serum antigen.
This conclusion was like a cold shower for us. Indeed, our antigen was a serum protein. It could be synthesized in liver and/or some other organs in response to tumor growth; it could be one of the acute phase proteins, which were already known at that time.
These proteins were synthesized in liver and were released into the blood stream in response to various pathological conditions. For example, a similar protein was detected in blood of rats in response to growth of various tumors. Appearance of such protein would represent a nonspecific response of an organism to various pathological processes. After appearance in blood such protein could be transported to various organs and accumulated in tumors and necrotic tissues. Although we washed tumors free from blood, its traces would surely remain anyway, so that this protein might just represent contamination unrelated to the tumor.
We analyzed blood of mice bearing hepatoma-22a and found very high levels of this antigen. Interestingly, blood levels were much higher than that of tumor; this antigen was also detected in organs of hepatoma-bearing animals. In previous experiments we used only normal organs of healthy animals, and this explained why we did not find our antigen there. It became clear that we were dealing with acute phase protein, which we considered as a tumor-specific antigen. If this suggestion were correct, this antigen would be formed in organisms in response to any tumor growth, say sarcoma, which is unrelated to hepatoma.
So we went to Olga Lezhneva, a member of Zilber's laboratory who was working with mice transplanted with carcinogenic sarcomas. We took blood from these animals and analyzed these samples for precipitation. The next day we detected the presence of our «hepatoma antigen» in one of four investigated samples.
The worst suggestion was almost confirmed. The only hope was that one of the blood samples was taken from a pregnant mouse. (Since embryo contains huge amounts of this antigen, it could be also detected in blood of the pregnant mouse.)
We immediately went to the animal house and found that there were newborn mice in the cage with our mice! But it was unclear which mouse gave birth to these pups!
So, we took blood from male mice transplanted with sarcomas and other tumors and analyzed blood samples for the presence of the hepatoma antigen. We did not find it, and this fact gave slight hope that our antigen was really associated with liver tumors.
We did not find this antigen in the blood of animals with other tumors. However, it might be possible that this antigen appears in blood only in response to hepatoma growth (or preferential hepatoma growth). To exclude all other possibilities, we had to demonstrate in direct experiments the synthesis of this antigen by our hepatoma. If we were to demonstrate this phenomenon, it would become clear that we were dealing with some embryonic antigen, which was reexpressed in liver cancer. This was very interesting. However, I should remind again that we had to demonstrate antigen synthesis by tumor.
It was a soluble (but difficult) task. We started to solve this problem using several approaches. These included heterotransplantation of hepatoma to hamster cheek pouch and to cortisone-pretreated rats. Our suggestion was that if mouse tumor synthesizes our antigen, we would find it in hamster blood and in rats. In parallel we started our joint experiments on the hepatoma antigen in tissue culture with Dr. I. S. Irlin and Ms. S. D. Perova, who joined our group when Mrs. Khramkova went for maternity
leave. We tried to get hepatoma primary cultures. If the antigen was synthesized in cell cultures, we would get the best and the most valuable evidence for its synthesis by tumor. We were quite lucky in these experiments.
First, we found some quantities of our antigen in the hamster cheek pouch. We did not get large tumors in the hamster pouch, there were just small nodules. However, we were able to detect the hepatoma antigen in these nodules and their extracts. It was possible that this antigen was synthesized by tumor. However, it was also possible that we had introduced it during transplantation.
In rats we got clearer results. In rats the mouse tumor had rapidly grown in two weeks and then it started to resolve. Thus, in parallel to the tumor development and resolution, we were able to detect our hepatoma antigen in blood, which disappeared during tumor resolution. However, some problems still remained: since mouse and rat are closely related species, do cross-reacting antigens exist? Using rat embryonic serum we found a cross-reacting antigen, exhibiting potent suppression of our test system for hepatoma antigen. (This suggested the existence of a related antigen.) Thus, we felt that results of these experiments suggested the existence of mouse antigen analog in rats, so we were still looking for additional evidence.
Two months passed and we finally obtained hepatoma primary cultures in vitro. In the culture medium we found the hepatoma antigen! We washed the cell culture to exclude possible blood contamination, we changed cultivation medium, but the antigen was detected. Thus it became clear that the hepatoma antigen had been synthesized in the tumor and that we caught an interesting phenomenon. In embryos this protein was probably synthesized in the liver as some serum protein, which we called af-globulin or fetal a-globulin. Embryonic concentrations of this af-globulin significantly exceeded the concentrations of serum albumin; however, during subsequent development synthesis of this antigen stopped and appeared again in primary liver cancer (hepatocellular carcinoma). Although this phenomenon was not observed in all the cases, it was definitely noted in rapidly grown anaplastic hepatomas rather than in highly differentiated hepatomas. Anyway, in most hepatomas synthesis of this antigen was resumed.
Hepatoma-22a used in our crucial experiments was the most potent producer of this a-globulin. So we were very lucky to use it in the very beginning of our studies!
Thus, it became clear that liver tumor might reexpress the embryonic antigen, which is almost totally suppressed in cells of adult liver. This meant that embryonic antigens specific for particular tissue might appear again in tumors derived from this particular tissue. Moreover, these antigens might appear not only in the tumor but also they could be secreted into blood (provided that this is a secretory protein). Consequently, an embryonic antigen, which is absent in blood of healthy adult mice, may be a tumor marker detectable in tumor as well as in blood of tumorbearing animals. Later we found that this phenomenon is also typical in rats. Thus it became clear that this is a common phenomenon at least in the cases of mouse and rat hepatomas.
This story on the discovery of hepatoma antigen in embryonic liver began in January 1962, and in July 1962 the 8th World Cancer Congress would be held in Moscow. Our lecture was scheduled into its program.
Here I should say that Professor Peter Grabar, a distinguished French immunochemist, the author of the method of immunoelectrophoresis, was familiar with results of our previous studies on the hepatoma antigen. Being interested in this problem, he invited me to report our results at a so-called panel discussion of the congress because this was the first tumor antigen isolated using immunochemical methods. We sent an abstract of our presentation before our discovery that this is the embryonic antigen: reexpression of the embryonic antigen described above became clear only in May 1962. The Gamaleya Institute was being refurbished: the administration was preparing the institute building to meet guests of the congress. We were constantly moved from our laboratory rooms, and I still do not understand how we were able to clarify the situation with our antigen within four months. Anyway, in July 1962 I reported our results at the 8th Cancer Congress. The audience appreciated our results, however, discussion went in several directions. Most questions were focused on the problem of the existence of specific hepatoma antigen (besides the embryonic one). Shortly after the congress our results became available for the Soviet and international scientific audience [8,9].
A very important continuation of the above data was obtained by Yu. S. Tatarinov, a biochemist from Astrakhan who was interested in human serum proteins in hepatic pathologies. We had known each other for about two years, and he already started to characterize the proteins in embryonic serum. Although it was clear that such system might have diagnostic value, we did not push forward such a project. However, some results indicated that our antigen would be a marker of hepatocyte proliferation, because its temporary synthesis was observed in mouse regenerating liver. We regard this antigen as the marker of hepatocyte proliferation rather than the immunodiagnostic marker of hepatic cancer. So, we were not inspired with the idea of clinical studies in humans. Tatarinov obtained corresponding anti-embryonic serum, absorbed it with adult serum, and then analyzed the serum obtained from a hepatoma patient. In the very first experiments he obtained convincing evidence for the presence of embryonic a globulin in it. Thus it became clear that the described phenomenon also exists in humans.
In the beginning of 1964 Tatarinov reported his data at the First All-Union Biochemical Congress in Leningrad and published a paper in the journal «Voprosy Meditsinskoi Khimii». Thus, he demonstrated an important diagnostic aspect of this problem.
I always appreciated (and appreciate now) Tatarinov's priority in the diagnostic aspect. His work in Astrakhan was much more difficult than ours in Moscow, in Zilber's laboratory. So I «restrained» activity of my coworkers trying to get into liver cancer diagnostics and reserved this area for Tatarinov.
However, in 1965 or 1966, Dr. N. I. Perevodchikova, a clinician from the Institute of Experimental and Clinical Oncology, asked me to study embryonic a-globulin in hepatic cancer for evaluation of its possible diagnostic value. I advised her to contact Tatarinov, but she desperately wanted to work with us. Tatarinov had already described six clinical cases and his priority in this problem was unquestioned anyway. In my papers I always cited his works.
However, I was a bit skeptical about the diagnostic value of the embryonic a-globulin, and so we started to work with Dr. Perevodchikova. Quite soon we confirmed Tatarinov's observation on appearance of af-globulin in blood of patients with primary hepatic cancer. However, we found its appearance in only 70% of cases. Furthermore, we also demonstrated appearance of af-globulin in blood of patients with testicular teratoblastomas. This was very important because it became clear that this protein appeared in tumors originating from embryonic cells. The reason for appearance of af-globulin in the embryonic cell-derived tumors was unclear.
Subsequent studies revealed that this was as important as the diagnostic aspect for primary liver cancer. In 1966–1967 we obtained a more or less clear picture using representative clinical material obtained from 55 patients with primary liver cancer and many patients with testicular teratoblastoma. We published results of our observations in the International Journal of Cancer [10], and this paper was appreciated by the international scientific community, especially by tumor immunologists. Together with Tatarinov's papers, it basically opened a diagnostic aspect of this problem [11]. It became clear that embryonic antigens may appear in a tumor, be released into blood, and they may serve as diagnostic markers of tumor(s). Thus, embryonic antigens may be used for specific serologic diagnostics of cancer, for its differential diagnostics [12,13]. Soon after our first publications on hepatoma, Gold and Freedman (1965) referring to our works and using our approaches (including the method of precipitation in agar and the quadrant method) detected specific antigens in colorectal tumors, and then they also demonstrated the embryonic nature of those antigens In 1969 these authors also demonstrated that low concentrations of the carcino-embryonic antigen (CEA) could be detected in blood of patients with colorectal cancer and might serve as a diagnostic marker.
Schematic representation of alpha-fetoprotein (AFP) synthesis in normal development and pathologic states. Solid line, serum AFP level in arbitrary units; broken line, expected AFP level; ?, not known. From Ref. [12] |
In the second half of the 1960s it became clear that embryonic antigens might appear in various tumors. These antigens may be secreted into blood or appear in blood via other routes. Their detection in blood is a diagnostic sign of a particular form of cancer. Subsequent works by our laboratory and by Czech scientists revealed that the time-course of these antigens in blood well corresponded to the time-course of development of corresponding tumors. In the end of the 1960s, there were three tumor types that could be diagnosed by embryonic antigens: primary liver cancer, testicular teratoblastoma, and colorectal cancer.
Resonance and Subsequent Studies
We started to publish results of our studies in 1959; our key paper on embryonic antigen in hepatomas appeared in 1963 and attracted much attention from the scientific community. However, this interest enormously increased after elucidation of diagnostic aspects of this problem (i.e. from 1967). This international interest in our work was also stimulated by Professor Grabar, who was very familiar with our results. His own laboratory in the Villejuif Institute for Scientific Research on Cancer in France was involved in studies of related problems, and starting in 1965 they obtained results that were consistent with our results obtained in rats. Grabar and his colleagues also started to develop the diagnostic aspect of this problem. They established good contacts with Professor Rene Masseyeff, who worked in Dakar University (Senegal) at that time. Owing to high frequency of primary liver cancer in West Africa, they collected a large amount of material supporting our first observations. Thus, interest in the problem increased. My contacts with Howard Goodman, Head of the Immunology Unit at the World Health Organization and a very nice and intelligent man, resulted in arrangement of an international experiment in African countries on validation of a diagnostic value of af-globulin. This experiment started in 1968 with our trip with Tatarinov to research centers of African countries involved in studies of primary liver cancer. This was a very impressive trip with adventures, demonstration, and propaganda for our results, with establishment of new, close, and open scientific contacts.
Finally, our activity culminated in establishment of international experiments, which gave very clear positive results in 1969; they were published in 1970 in «Cancer» [14].
All these events resulted in rapid international recognition of the alpha-fetoprotein (AFP) test and its wide distribution in all countries. The AFP test was applied also in cases of testicular and ovarian teratoblastomas, where it could evaluate effectiveness of medical treatment. In contrast to primary liver cancer, these tumors can be effectively treated by surgery and chemotherapy and therefore evaluation of effectiveness of such treatment by a simple serologic test is very important.
In the end of the 1970s, the total number of publications on AFP exceeded two thousand, and this problem smoothly entered both science and clinical medicine without hot discussions or skeptical resistance. Of course, there were some disputes with Jose Uriel, Grabar's pupil, but they were devoted to some «local aspects», and we soon came to a common viewpoint and maintained friendly relations. In 1975 this work received one of the First Prizes on Cancer Immunology awarded by the New York Cancer Research Institute.
In the very beginning of the 1970s, Japanese Professor Hidematsu Hirai organized an international group for studies of cancer embryonic proteins. In 1980 this group transformed into the International Society for Oncodevelopmental Biology and Medi-cine (ISOBM). In 1976 I received a prize awarded by this group for discovery of AFP in hepatoma. Earlier (in 1973) I was elected as an Honorary Member of the American Association of Immunologists. I was very pleased with these awards. (I should say that I even did not know that I had been nominated for these awards.) In 1978, together with Tatarinov, we received the USSR State Prize for the discovery and practical application of AFP in cancer. We received this prize on the «third attempt». In 1971, the Gamaleya Institute nominated us together with a group of colleagues for the State Prize, but our work was rejected. The same situation was repeated in 1976. Finally, working in the USSR Cancer Research Center, we were nominated for the State Prize and received it in 1978. In 1990 I received the Abbot Prize awarded by the International Society for Oncodevelopmental Biology and Medicine. Although the series of our studies were internationally recognized and received prestigious prizes and awards, this period of international recognition coincided with a very hard time in my life. In 1966, L. A. Zilber suddenly passed away, and all members of his department suggested me as the new Head. The administration of Gamaleya Institute and its Director, Academician O. V. Baroyan of the USSR Academy of Medical Sciences, had to accept me as the new head of Zilber's department. For three years I worked as Executive Head of this department, and in 1969 I was elected to this position. Everything looked fine: studies successfully went in the directions we wanted. In 1971 we developed a diagnostic kit for AFP and gave it to the production department at the Gamaleya Institute. This was the first kit for immunodiagnostics of cancer in the world. During that period I was in frequent contacts with the World Health Organization, Institute for Scientific Research on Cancer in France, and often traveled abroad. However, in 1971 everything changed.
In 1971 the first people who desperately wanted legal emigration to Israel appeared. They were blamed by formal Soviet public opinion and treated as «traitors to the fatherland». Scientists lost their jobs, and they could not get necessary documents required for emigration visas. Laboratories where such people worked had serious problems as well. Waiting for official permission for emigration varied from several months up to many years without any reasonable explanation. The presence of such persons among staff members of the institute and especially the laboratory represented basically «nightmares» for administrators. Usually dismissal of such potential emigrants occurred after public discussion of their personalities, which always ended by very rough blaming of «such antisocial persons». People with Jewish background who remained loyal to the Soviet regime had to participate actively in such a «farce», irrespectively to their own viewpoints.
In the end of 1971 one microbiologist, a junior researcher of the institute, decided to emigrate to Israel. At the forthcoming meeting of the Scientific Board at Gamaleya Institute, this person would be reelected to his position. (This is a common practice in all research institutes.) However, in reality he could not be reelected for a new period. The real goal of that meeting was to blame that potential emigrant. All heads of laboratories with Jewish background had to participate in that meeting. Besides blaming the «renegade», the administration expected that Jews of our institute would demonstrate their own loyalty. I was not going to emigrate, but I did not want to play a role in such a shameful farce either. Although I was a member of the Scientific Board, I ignored that meeting.
My «demarche» caused almost immediate and hard reaction from a director of the Gamaleya Institute. He slightly changed the names of two laboratories of Zilber's department, but this caused serious consequences. My laboratory, the Laboratory of Immunochemistry, was renamed as Laboratory of Immunochemistry and Diagnostics of Cancer. The other one, the Laboratory of Cancer Virology, was renamed as Laboratory of Viral Etiology of Cancer. This formal «reorganization» resulted in dismissal of all coworkers, and we had to apply for a position in these «new» laboratories, as all scientists who should be elected anew.
At the same time, the scientific community of Moscow supported us very actively. Academicians Vladimir Engelhardt and Boris Astaurov, directors of the Molecular Biology (V. E.) and Developmental Biology (B. A.) Institutes, respectively, called a conference in the academy devoted to cancer virology and immunology and asked me to present the results of our work.
Our director appointed a special meeting of the institute scientific board with my account on the work of my laboratory just on the same day and at the same time, so that I could not do my presentation in the academic meeting called by Engelhardt and Astaurov.
The only way to join this meeting was to retire from the work in my institute. I did so, made the presentation at the academic meeting, lost the work in the institute, got positive support from the scientific community of Moscow, and started the struggle for the transfer of my laboratory to the Oncology Institute. I asked the president of the Academy of Medical Sciences to transfer our laboratory into the Oncology Institute.
My colleagues and I protested against this despotism and the director (an energetic, hard, and severe man) initiated rough «strikebreaking». Echo of our «war» with him reached the President and the Presidium of the Academy of Medical Sciences. The Presidium formally took our side and liquidated the «fire of this war». However, this victory left «painful scars» in my soul. I should say that I have never had enemies and I do not have them now (both in scientific and human terms). Nevertheless, from time to time I am involved in conflicts with state and institute administrations. I think that such conflicts originate from my subconscious resistance to humiliations. Anyway, that «war of 1971» «burned» us. Of course we continued our work, but the whole team of our laboratory was disturbed. We felt that we were «under the gun-sight» of the administration of the Gamaleya Institute, and nobody inside the institute could help us (although many people did sympathize with us silently). This was a very difficult period of my life. My scientific visits abroad immediately ceased. I could not attend even conferences on AFP or cancer immunodiagnostics. Our work was completely ignored by the administration. Literally speaking, we had to keep «circle defense». This situation coincided with serious changes in directions in analytical and applied studies on AFP. At that moment I started 37 my fight for transfer of the department to Blokhin's Oncology Institute*. I thought that this would give us the possibility for normal and productive research.
* Now N. N. Blokhin Russian Oncological Scientific Center of the Russian Academy of Medical Sciences. |
Finally, in 1977 this fight with the administration of the Gamaleya Institute finished by our movement to the Oncology Institute. During these six years we worked very intensively (as intensively as before), but we were almost fully isolated from the international scientific community. Nevertheless, this was a very fruitful period, which was also appreciated by our colleagues. I published a review, one of the first reviews on alpha-fetoprotein [12], which was recognized as a Citation Classic by «Current Contents» (800 references during 1971–1980). The other work (by N. V. Engelhardt, A. K. Yazova, and V. S. Poltoranina) underlined reasons for synthesis of AFP by experimental testicular and ovarian teratoblastomas [15]. This work demonstrated that this protein was synthesized in germinal tumors because of development of the element of embryonic yolk sac. (Besides liver embryonic yolk sac, entoderm also synthesizes AFP in normal ontogenesis as well.) This was a very nice and exact work.
The third work of that period was related to the development of a highly sensitive method for determination of not only AFP, but other antigens as well; this method was based on isotachophoresis in polyacrylamide gel. This method demonstrated the presence of traces of AFP in sera of normal animals and revealed the basic level of this protein in serum of healthy people. This study was subsequently confirmed by others. In collaboration with S. D. Perova, we developed a very nice, precise, and elegant variant of the method for determination of AFP synthesis by single cells of hepatoma or hepatocytes in a cell culture or in micro-colonies of these cells [16]. During that period, we (A. I. Gusev, A. K. Yazova, and S. D. Perova) also developed a commercial version of an immunodiagnostic kit for simple, convenient, and inexpensive AFP assay, which was produced by the Gamaleya Institute for about 15 years.
Finally, there was one polemic paper, which I prepared together with my first (late) wife as a response (or maybe as our reaction) to all these events in our lives. Besides my resistance to the above-mentioned administrative pressure, we were writing that paper about ethics, about the role of ethics in science. This paper was «our manifesto» about normal human relations in science and our personal viewpoint about organization and administration in science. Literally speaking, that paper was written with our blood. We sent the manuscript to the Soviet journal «Nature» («Priroda»), where it had been positively reviewed by Academician V. A. Engelhardt, but in spite of this it was not published. Quite unexpectedly, it appeared in another journal, Chemistry and Life, in the beginning of 1985 [17]. Thus, 13 years later this paper was published in slightly a shortened variant, but our major ideas are preserved there. (I should say that after publication of our paper, this journal had serious problems.)
In spite of all attendant circumstances, we continued our research. We were involved in studies of a new interesting erythroblast antigen, which we intensively studied and which had clinical application as well [18]. We also studied the problem of expression of endogenous viral antigens [19] during normal mouse development and in tumors. Our investigation of morphological aspects of AFP during liver 15 regeneration represented a basis for subsequent studies. Our main problem was that of AFP regulation: why does AFP 17 cease to be produced in liver maturation, and why it is reexpressed in hepatocellular carcinomas?
The main observation important for understanding of this problem was the role of cell interaction in the suppression of AFP synthesis. It was shown that AFP synthesis is resumed after poisoning of the liver by hepatotoxins in the hepatocytes bordering the necrotic area. These cells lost their contacts with neighbors and seemed to be isolated from the liver plate [20]. The isolation of hepatocytes from the adult liver by treatment with collagenase in Ca2+-free medium led to AFP production in the overwhelming majority of cells. Restitution of intercellular contacts suppressed AFP synthesis, while inclusion of hepatocytes in a three-dimensional extracellular matrix resulted in formation of liver-like islands with reestablishment of cell polarity, bile canaliculi, and full suppression of AFP [21].
The cell–extracellular matrix interaction in three-dimensions became the major factor in induction and maintenance of hepatocytes differentiation, including suppression of AFP [22]. This could be the reason for AFP reexpression in liver tumors. This is the problem of today's and tomorrow's research.
Concluding Remarks
My opinion on the significance of our work. I should say that our studies leading to discovery of AFP in hepatomas were our most important works. On the one hand, they represented a basis for the development of the «carcino-embryonic» direction in oncology, and on the other they started cancer immunodiagnostics.
Very important for us is the study of regulation of AFP synthesis in liver development and regeneration.
However, in my very personal viewpoint, our studies of wide expression of the endogenous retroviral antigen are also very attractive and important. They basically represented the first immunologic evidence for the ubiquitous distribution of endogenous retroviruses in the mouse genome. We investigated the expression of this viral genome in mouse ontogenesis. I still like this work of 1970, and do believe that it is a very important study. This work was also appreciated by the scientific community, but to a much lesser extent compared with our studies on AFP.
I like my early series of papers on separators, especially on the chamber separator. This was an original device of new construction in pair with a fully original method of particle separation both by dimension and density. Although these works were appreciated and recognized, there was no further development of this approach in other laboratories because of lack of laboratory separator production and commercial realization of these devices. This is very annoying and disappointing.
I like my work on the method of immunofiltration that ensures isolation and purification of antigens specific for a certain particular system. I do not say that this work did not have scientific resonance. It did. And from time to time similar methods appear in various modifications. Finally, I like my work of 1979. It gave a push to a subsequent series of studies on counterflow isotachophoresis in porous membranes. I feel that in that work we found a new principle of electrophoresis, its new version: flow isoelectric focusing based on automatic isotachophoresis, rather than the specific electrophoretic method. This work required intellectual efforts and accurate and long-term analysis of very simple and unclear phenomena, which take place in hydrophilic porous membranes during electrophoresis.
Suddenly, I understood the reasons for abnormal behavior of liquid in the membrane during electrophoresis. This understanding resulted in the development of a flexible method with wide capacities. Its use allows simultaneous concentrating and effective separation of proteins from a solution, which is in flow and contains trace amounts of these proteins. The lesser the concentration of protein the more effective the method is (although the method itself is rather simple and theoretically clear) [23]. This method was employed for diagnostics of malignant B-lymphomas using urinary proteins. I think this method has promise in nephrology, in obstetrics, basically everywhere that analysis of protein composition in biological liquids with low-protein content is required. Using this method we developed a completely new principle of automated multistage immunochemical reactions and a new arsenal of methods for work with monoclonal antibodies (from screening of monoclonal antibodies and including epitope analysis) [24]. Thus, certain manipulations involving monoclonal antibodies can be transformed in automated mode of analyses based on counterflow isotachophoresis in porous membranes. Although we employed this method for several years and have published it in Russian and international journals, its resonance is rather small. We know advantages of this method and its large capabilities, but it is still in a latent period. It is possible that this situation occurs due to popularity of methods that require commercially available reagents produced by special companies. Possibly this happens because of rather bulky description of this method; the method itself is very simple, and I do not understand why other laboratories do not use it. At least we would like to see it being employed by others.
However, time goes fast and each method is good for its particular time. It is really sad that during many years this method has been used mainly in our laboratory; it would be very useful in many other studies.
Some formal information. My first scientific paper was published in 1953. It dealt with the method of sample preparation for electron microscopy from saline solutions. I still like this paper; it is very short and simple. I was involved in electron microscopy in Zilber's laboratory; he wanted me to use electron microscopy, but I did not like it. Anyway, I developed a method for electron microscopy of samples avoiding distilled water, a stage required for salt removal [25]. However, three years earlier A. N. Belozersky and N. V. Proskuryakov published their textbook Practical Works in Plant Biochemistry (1950) with some modifications of the methods, which I developed in my diploma project. They described these methods and acknowledged my authorship, and I was very proud.
I obtained my Ph.D. degree in 1955 and my life changed. First of all my salary increased, and this was very important because I had a family with two children. Right after defense of my Ph.D. thesis I finished all my extra works, which I needed to get money.
(I used to write abstracts, worked as a teacher, and even as a loader.) After public defense of my Ph.D. thesis I worked only as a researcher.
In 1963 I defended my D.Sci. thesis devoted to identification of individual tumor antigens. It included all materials on gel precipitation, all methodical materials related to immunofiltration, isolation, and purification of tumor-specific antigen; and initial results of studies of the embryonic nature of this antigen.
I should say that I did not write my thesis; my defense of the D.Sci. degree was based on a monograph written together with Zilber [3]. It was an interesting story. Zilber had a contract with Medgiz (State Publishing House for Medical Literature). According to this contract he had to write a book on cancer virology and immunology by 1961. Zilber was very busy and he could not finish that book by the time indicated in the contract. It was especially difficult for him to write chapters devoted to analysis of recent immunogenetic studies. During that period he was especially interested in cancer virology, and he did not have enough time for chapters on immunogenetics and histocompatibility antigens.
This particularly important direction was just begun to develop very intensively. Zilber asked me to analyze the literature on these subjects and to write a chapter to his book. I readily started this work, entered the problem, and analyzed unusual and complicated literature. I wrote a chapter on transplantation antigens, tissue compatibility, and its genetic control and also on the role of the transplantation antigens and their importance for studies of cancer immunology. Zilber liked this chapter and he invited me to write one more chapter, and then other chaptersЕ Basically I wrote almost the whole section of that book devoted to cancer immunology, which represented more than half of that book. This section contained results obtained by Zilber's and our team. (Our laboratory was just being organized.) At that time we did not know that the hepatoma antigen is the embryonic antigen. This book was finished in 1961 just before the discovery of AFP, and it also included all methods that we developed. This explains why I did not want to «copy» the same material for a thesis. It was a nice liberal time in Soviet science when it was believed that scientists should do good research, whereas the form of its presentation is less important. In 1963 I defended my D.Sci. degree using the immunological section of that book, which included all data before AFP and the very beginning of alpha-fetoprotein re-search. My defense of the D.sci. degree was quite successful in the Academy of Medical Sciences of the USSR; however, problems began when all documents were sent to the High Attestation Committee. (This is usual practice of Soviet and Russian science: results of public defense of Ph.D. and D.Sci. theses have to be approved by the High Attestation Committee.) One referee of that committee concluded that I had to write a whole text and repeat the whole public defense again. Fortunately, the other referee wrote a very positive report about «my part» of the book and in 1963 the High Attestation Committee approved the results of my public defense, and thus I got my D.Sci. degree.
The book by Zilber and me, published in 1962, was translated and edited by Pergamon Press in 1968.
All my younger colleagues (N. V. Engelhardt, N. I. Khramkova, A. I. Gusev, V. S. Tsvetkov, and T. D. Beloshapkina) defended their Ph.D. theses in 1964–1967, and in 1967 I received the title of Professor. In 1964 a new important period began in my scientific life: I started to give lectures on immunochemistry at the Chair of Virology of Moscow State University organized by Professor A. N. Belozersky. I was among the first invited pedagogues at this chair. For this cycle of lectures, I had to «translate» medical problems of immunology into common «biological» language. I wanted to demonstrate to my students that immunology is a biological science, which examines problems of cell differentiation, genetic control of protein synthesis, employing unique biological models. Under certain circumstances these models underline basic biological processes even better than classic developmental biology models. I wanted to introduce immunology for university students in a way that would be interesting for them from the biological viewpoint. However, I also wanted to demonstrate to them that biological problems of immunology represent a basis that may be used for understanding of pathogenesis of many diseases and for understanding of common medical problems such as inflammation or allergy. This task, the introduction of this medical science for «purely biological students», was very interesting, and I surrendered wholly to this work. I think that my course in immunology was interesting for the university students. I still give a cycle of lectures in immunology (2005) using the same principles, but of course using modern scientific level and material.
I think that my personal self-evaluation as a professional expert in a particular field of immunochemistry appeared after mastering the method of immunodiffusion (and related subjects).
We became very familiar with all the delicate details of this method, and my confidence in this particular field extrapolated to other things. This was very typical for me. I always extended frameworks of my profession or alternatively in attempts to solve a problem I entered in a new field, which required completely new competence in this new field. Basically, the race of problem solution constantly exceeds limits of my competence. In the very beginning of my life in science I left biochemistry for immunochemistry, then from analytical immunochemistry I moved to immunomorphology and cell biology, but then I came back to electrophoresis. Thus, my search for a solution to particular scientific problems is accompanied by partial loss of previous competence. I think that a specialization of a scientist trying to solve a scientific problem is the specialization on resolution of non-standard problems. This requires a certain kind of confidence, which should be combined with a strong diffidence. Such combination of self-confidence and diffidence accompanies all my life in science.
I do not know whether this is my personal characteristic feature or a professional feature of real scientists. I prefer to think that this is a professional feature as well. One cannot be a real researcher if one pre-limits one's own professional competence and professional confidence. Real scientific problem always overcomes these limits. Thus, the major question is should one follow the problem, when it leaves predetermined professional limits, or not? For me there was not a question: I always left areas of my own competence.
Conflicts of ideas or viewpoints. I do not remember actual conflicts as such. There were some conflicts of ideas, especially when Zilber was alive. I respected Zilber very much. He was an extraordinary, outstanding man. Zilber was «a large scale» man of principles, very active, very impatient. He believed that a scientist should work only on the resolution of important principal problems. He used to say that it is absolutely right, because work 5 on principal and «second-best» problems is equally hard, or at least they require comparable efforts and time. Consequently, it is better to be involved in works on principal problems. He always followed this rule. Works on «second-best» problems were inconsistent with his nature. Zilber often said that it is impossible to «sew the last button on a work»; basically he did not like to lead research up to a refined state. He preferred to resolve problems in general and to leave details for others. He always appreciated «large and wide» problems. He had a wonderful feature: he never had inertia of previous success, and he was able to change scientific problems and research areas. This was a great feature, which helped Zilber to make significant changes in his life and important independent discoveries.
When we had demonstrated the presence of embryonic antigen in tumor he said: «Enough. You have made a good job and now apply your experience and your systems and approaches for recognition of specific tumor antigens. Embryonic antigen is interesting for a narrow group of researchers, whereas specific antigens are a really hot spot, which would be interesting for all. It will definitely change the whole of oncology». In his intention to persuade us to do most important things, Zilber refused to sign our requests for laboratory mice, which we needed for this work.
However, for my colleagues and me, it was impossible to leave this work unfinished. I thought that if study was not clarified all crucial points, it should be continued. Really completed work, its results begin «its life» after elucidation of precise mechanisms of phenomenon and putative consequences. I continued my work in spite of completely different viewpoint of Zilber and would not leave it unfinished. Zilber respected me, I deeply respected him, and therefore conflicts of our ideas cannot be classified as struggle. However, I should say that such conflicts of ideas were quite frequent events.
Now I should say that we both were not right at that time. We did not think about diagnostic aspects of this problem. However, subsequent development of liver cancer and germ cell tumor diagnostics originated from the discoveries associated with embryonic antigens. Anyway, this was one of the first results of basic studies that were introduced into clinical practice and gave clear clinical results. This perfectly fitted Zilber's concept of introducing basic results into practice.
I did not have real scientific opponents. Results of our work and our viewpoints were readily accepted and reproduced by scientists in different laboratories without resistance or scientific struggle. I think that there is an overestimated notion about our laboratory. This may be due to our position: we do not develop eccentric super-original concepts; we always set experiments before theoretical conclusions, which are always based on results of our experiments. Anyway, I should say that I have not met scientific opponents either in the literature or in discussions. From time to time I wage war with administrators. However, my wars are related to organization of scientific process, but not to science itself.
The only things that I strongly resist since the beginning of the 1970s are hierarchical principle of organization of science, monopoly, and administration in science. I struggled for these principles in the past and I struggle for them now. I think that this is my primary scientific duty.
I am interested in a wide spectrum of studies in my field and related areas. These include research on general immunology, T-cell receptors, and intercellular interactions. I am interested in these studies from both scientific and pedagogical viewpoints. (I always renew my lecture material for my students.) My current professional interest includes methods in immunochemistry, problems related to differentiation of tumors, immunodiagnostics of cancer and leukemias, problems in tumor biology, regulation of AFP synthesis, etc.
Among studies by Russian scientists, I like to mention interesting works by the late A. Ya. Fridenstein on the microenvironment and differentiation of stem cells. He used original approaches and ideas on the differentiation of stem cells, which influenced me very much. I am interested in studies by Yu. M. Vasiliev on the role of extracellular matrix in regulation of specific biosyntheses. These works are directly related to our studies. Interesting results on immunology of metastasizing have been carried out by G. I. Deichman. I should also mention papers by A. D. Altstein on construction of antiviral vaccines that may serve as antitumor drugs.
It is difficult to say which contemporary scientists–immunologists are authoritative for me now. Twenty years ago I might mention M. Burnett, G. Nossal, P. Medavar, and M. Hasek. Now it is difficult for me to select outstanding contemporary scientists.
There are many «first class» studies and brilliant scientists, and so I am not confident that I can choose outstanding scientists determining current progress in immunology.
Nevertheless, I want to mention some very distinguished experts in my field. These are George Klein (virologist, immunologist, oncologist), Cesar Milstein and Georg Köhler (they developed hybridoma technology), Lloyd Old and Edward Boys (among founders of cancer immunology, making important contribution to development of this field), and Susumu Tonegawa, who demonstrated genetic recombination in the production of antibodies for the first time.
I have already written about my teachers, especially about my Moscow State University teachers. My reader clearly understands the particular role Zilber had in my life. I hope that everyone who reads this paper realizes that Zilber played a decisive role in my choice of research field where I am still working, on selection of scientific problems, and research work as well. His strongest influence on me consists in the following: he never let us (his co-workers) work on local problems. Sometimes he even forced us to leave them and to focus our attention on global problems. I lost this inestimable influence on my research with his death.
Zilber was characterized by a wonderful feature, special width in his approaches to scientific and human problems. This width was «his intrinsic property». He could cover the whole particular problem starting from its background, monitor its development, and analyze its relation to adjacent areas. I always liked this feature of Zilber's and I tried to inherit it from him.
There are several members in my team, who jointed our laboratory for a diploma project or as graduates from the university. Now they are mature independent scientists with their own names in science. I do not know whether they consider me as their teacher. However, I taught them to elaborate and follow some criteria of their scientific work: tolerance, reliability, and reproducibility. I do not say that there were special lessons. Things came naturally during experiments and in the process of discussion of their results, when we elaborated some general criteria in evaluation of our work, its merit, originality, and mainly reliability. It is possible that this is the inherent style of my students.
Generally I tended to develop their own individuality in my students and junior colleagues. In some cases I achieved my goals.
I treat my colleagues very attentively and possibly I know their strongest sides better than they themselves. I always wanted that my younger colleagues should develop their best sides themselves.
In people I especially like those features that I do not have myself, and I never suppress individuality of my younger colleagues.
Moreover, working together I always try to identify «genes underlying their personalities», their inclinations, capacities, and interests. I do believe that the learning process is the process of «purification» of one's own interests and talent. All my students are very different and each of them has their own individual «face».
A. S. Gleiberman can be readily provoked on a new project; he has a perfect flair on novelty. A. I. Gusev is a brilliant bench worker, S. D. Perova is an expert of biological experimentation, whereas N. V. Engelhardt is a perfect immunomorphologist. Each of my students is unique, but they all share one common feature, absolute intolerance to negligence, approximation, and peacockery. They completely differ from me, we are often interested in different things, we are different personalities, but we easily come to terms.
Twenty years ago scientists were subdivided into several «schools». There was Zilber's school, Shabad's school. However, this subdivision was typical for the «pre-paradigm period». Now, after formation of paradigms in cancer virology, cancer immunology, general immunology, I do not even know whether certain
scientific schools joining groups of like-minded persons actually exist. I think that in our field number of such schools gradually decreases because of structuring of our knowledge. In my viewpoint the schools now reflect some structural scientific organizations: laboratories, departments maintaining steady groups of scientists working together for many years. Such groups share
similarity in viewpoints on the investigated problems, criteria of success of their research. Perhaps this is the modern meaning of what we call the «scientific school» now. In my viewpoint borders between various schools of immunologists and virologists are spread out and schools themselves are «dissolved» into the general structure of current knowledge of a particular field. I myself was a student of Belozersky's school, belonged to Zilber's school. These schools influenced my subsequent activity. However, now I do not belong to any school.
I think that a young person, who wants to become a scientist, should have natural and potent interest in science; this interest should be one's own interest rather than reflection of fashion or ambitions. Real scientist should believe in their own interest in science, should follow it, and keep eyes and ears open. I think that a young scientist needs success to obtain confidence required for subsequent studies.
Real interest in science together with keenness on research results in such «fusion» with an investigated problem, which helps better understanding of hot spots of these particular problems and creates a good background for intuition. The latter is ultimately required for success of any investigation. Such pre-conditions are very important for a sketch of an internal image of the research object and when logics ends the scientist will intuitively find the route, allowing to see a «light at the end of the tunnel» and thus solve problem and understand truth. Secondary stimuli, such as ambitions and profit, cannot develop intuition in scientists and deep understanding of research object or problem and if these are the only (or leading) stimuli it is impossible to get real success in science.
The other important thing consists in analysis of particular problems. It is not right to pre-classify problems into primary and secondary ones. Scientists should solve problems, which they selected by themselves, and then «God decides» which positions of these solved problems will occupy the common tree of cognition. Anyway, I do believe that artificial selection of global or «nodal' problems, which bode success is a wrong way leading to a dead-end. The thing is that the problems which may be formulated as promising ones may be defined as the pre-resolved problems because they have been already formulated and pathways for their solution are more or less clear. In my viewpoint scientists should work on unresolvable tasks, originating from the own particular interest of these scientists.
I should say that I am happy with my life in science. Some results of our work played a certain role in immunology and oncology, some results still influence these fields. This gives me a sense of great satisfaction, because normally the net efficiency coefficient in science is very low. The major proportion of studies finishes at a dead-end without public resonance and subsequent development in other laboratories. Thus, I am pleased that at least some results of our studies have been recognized by the scientific community and received further development both in theory and practice.
All my life I have been working only in accordance with my own research interest. Although my way in science has not been straight, I have always followed my interest, the way that it has driven. I almost physically could not work on uninteresting things. However, I always worked with pleasant people, with which I felt quite comfortable. It is my constant belief that one should maintain only those scientific contacts that are quite nice and pleasant and also to work with those people who do not «spoil» comfortable, creative, and productive atmosphere in the lab.
The most successful and creative periods of my scientific carrier were in the middle of the 1950s (work of new construction of separators); the end of the 1950s and the beginning of the 1960s (works on immunodiffussion, immunofiltration, isolation of hepatoma antigen, and elucidation of its nature); the beginning and middle of the 1970s (the development of highly sensitive methods, study of endogenous viral antigens, the development of the method of counterflow isotachophoresis on porous membranes); the end of the 1970s and beginning of the 1980s (a burst of my engineering activity), and finally – the study of cell-extracellular matrix interaction as a critical step in liver cell differentiation.
In conclusion I want to say that in spite of successful and creative life in science I am not a perfectly satisfied scientist. This lack of «complete satisfaction» accompanies me during all my life. It is still with me. However, I feel that this is a normal state of a real researcher.
ACKNOWLEDGMENTS
I would like to express my sincere thanks to my wife, Galina Deichman, for constant assistance in the preparation of this chapter.
The help of professional translators Alexey Medvedev and Richard Lozier is greatly acknowledged.
My special thanks to Olga Salnikova for all technical work connected with the preparation and corrections of the manuscript.
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* Stories of Success Volume 45. Personal Recollections X. Edited by Giorgio Semenza. 2007 back