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ANNUAL PROGRESS REPORT Period: August 1, 1978 to May 1, 1979 Proj ect Title : Radiation and Chemical Effects on Viral Transformation and Tumor Antigen Expression University of South Alabama
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TABLE OF CONTENTS Background to Current Work ....... Work from this laboratory (1970-1977) Summary of recent results (1978-1979) Detailed report of recent work ...
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It is conservatively estimated that in excess of half the cancer experience in our population could be eliminated by restricting exposure t o environmental carcinogens(I). While such speculation seems t o have some of its roots in reality,we still need t o pursue methods for early cancer detection to effectively diagnose and hopefully develop means for eliminating those forms of cancer which will still occur i f our nation continues to be unable to control human exposure to carcinogens.Practically speaking,it seems impossible to avoid all carcinogens.Additionally,there i s mounting evidence that co - carcinogen es is may be involved in the induction of cancer,presenting a situation where i t may be extremely difficult to reliably assess carcinogenic hazard for decades t o come. An experimental case in point could be the studies we conducted i n 1969-1971. Radiation and radiomimetic chemicals markedly potenti a ted SV40 transformation' in vitro and oncogenesis in vivo in our studies(2,3). Neither the low radiation dose,the chemical concentration nor the virus dose employed individually would effectively produce transformation --i n vitro nor tumors i n animals, but in synergistic combinations were highly Tumor biology has become a perplexing field in the past twenty years. The burgeoning literature concerning transformation of cells and tumor immunology is rapidly forcing the conclusion that the diversity of findings and rise and fall of dogmas leave us with precious little that we can hold universally true about cancer cells or their intrinsic properties. Cells which exhibit many "normal" growth characteristics in vitro (e.g., 3T3 mouse fi br oblasts) can be oncogenic in vivo. Cells which are fully transformed -i n vitro will often not produce tumors i n vivo. The biochemical differences between tumor cells and normal cells seems completely open to conjecture in the main and little seems certain about the absolute significance of any specific chemical deviation in the cell or its membrane to the cancer state. Immunologic reactions detected vitro and directed against neo antigens on tumor cells by the host ' s immune system seem t o have no bearing on tumor cell survival in vivo. In excess of twenty immunologic methods were developed i n the past fifteen years which were touted to detect tumor specific immune reactivity. None has found its way into widespread, reliable use for the detection and diagnosis of cancer in man or animal with perhaps the single exception of the detection of alpha feto protein, and limitations exist even for this "marker" for liver cancer. The pattern of host immune reactivity to tumor is still confusing to interpret and meaningful generalities are still difficult to draw about the cancer state and how i t This is truly a depressing picture for the cancer patient and for the researcher when w e considered the great hope that immunology seemed to offer for cancer control ten years ago (see review by Bartlett, Kreider and Pur nell,reference 4). The picture is even more depressing when we realize that our society is moving further into an era of increasing environmental Since 1969 w e have been heavily involved ina search for characteristics of tumor cells which are universal,or so nearly so that the exceptions constitute a very small proportion of the to tat. Our search has been
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conducted primarily with tumors induced by viruses which are potenti a ted in their oncogenic action by low-level exposure tox - irradiation or to radiomimetic chemicals (e.g., 5-iododeoxyuridine). We observed that all tumors induced by DNA-containing, oncogenic viruses (SV40, adenovirus, polyoma) carried embryonic or fetal antigens in their plasma membrane spectrum of animal tumors induced by chemical carcinogens,radiation, DNA tumor viruses and those occurring spontaneously have been surveyed rather The general conclusion that the pr in cia pl investigator has been able to draw from these research efforts is that all tumors induced i n animals which have been rigorously examined have been observed to carry cross - reacting substances which can be detected by a variety of immunologic means. The cross - reactivity is his to logical y specific. Where carefully studied,these substances often represent re-expressed embryonic or fetal proteins(26). The apparent rare exceptions have only occurred among high passage tumor lines which have been carried extensively in vivo by artificial transplantation and which seem to carry no discern able transplantation Thus, after six years of study,we have become convinced the expression of these embryonic or fetal products (EA), which can act as auto antigens, may well be a universal characteristic of all cancer cells. Most major forms of human cancer carry cross - reacting substances within his to logic families and many of these are now recognized t o be embryonic-like in character (25). Considerable controversy has existed over the types of immune responses which embryonic substances, re-expressed on tumor cells, can stimulate (25,271, but it i s becoming strikingly clear that tumor cells universally carry the embryonic constituents and that they can be detected by one means We currently have sponsored support by the NCI to study the character of the 7h ost response t o embryonic antigens(EA) on SV40 tumors and t o attempt to distinguish EA from s o - called tumor specific transplantation antigens (TSTA) which also appeared on SV40 transformed cells. This project focuses on the role of these antigens in the immunologic reactions which can result i n tumor progression or of their possible use i n immune therapy against Under WE support this past year,we have attempted to utilize the immunologic tools, developed in the NCI sponsored contract and grant,to evaluate the early period of radiation stimulated tumor cell formation when sub-oncogenic virus concentrations were added t o normal hamster and human cells in vitro. Our search has been for an assay system which would allow us to detect the earliest time of expression of EA should these antigens be expressed early in radiation potenti a ted,viral oncogenesis. We had good data to suggest that the EA components were cryptic aly present i n normal adult cell membrane (25).Frankly, based on the disappointing data obtained in late 1974 and early1975, we anticipated that this would be the last year of this long-standing contract with DOE (ERDA, AEC) because of the extreme difficulty in meaningfully assaying normal cell populations infected with SV40 for early transform ants.All methods for detecting a small number of transformed cells among a large population of infected cells which had been tried to that time had been disappointing and suffered from large experimental deviation. These methods were ultimately dependent on cl on al
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colony format ion following subculture which did not permit ready evaluation of early transformed cells. This was true even though exposure of the normal target cells to 100-150 R of x-ray prior to infection with SV40 increased the transforming efficiency by 200-400 percent. It was clear from many of our studies that well-formed tumors stimulated by SY40 and potentiated by x-irradiation carried EA, but these cells could only be studied months after the original oncogenic event between radiation sensitized tumor cells and transforming virus. We needed to know more about the earliest transforming events if we were ever to be able to make meaningful determinations of specific changes in cells which reflected their conversion to transformed and subsequently to malignant cells. This basic information about early tumor cells is essential also if we are to ever "unravel" the mystery of the very complex immunologic responses which occur in the host experiencing the Then, in the fall of 1975, we made an intriguing observation, which opened the door for detailed immunologic analysis of early transformant cells induced by oncogenic viruses synergistically interacting with x-irradiation or chemicals which damage DNA and facilitate the integration of SV40 into host chromatin. We observed that shortly after the addition of SV40 to normal target cells in suspension culture a specific fraction of the cells begin to show affinity to attach to acrylamide surfaced in special cloning gels. Although the multiplicity of infection employed assured that all cells in the population are infected with several SV40 virions, the proportion of cells which attach to the acrylamide surface is very low, reflecting the usual pattern of one transformant per thousand cells which abortively harbor SV40. Normal fibroblasts do not attach and proliferate on acrylamide. Thus, we had a selective method for reliably obtaining and cloning early transformants. The ability to detect the transformants within a few hours after the oncogenic event(s) occurs affords many exciting possibilities for investigating immunologic transitions which occur and for exploring the molecular events involved in primary cell transformation The added advantage of being able to raise the transforming frequency by 400 percent by presensitizing the target cells to SV40 transformation with x-irradiation amplifies enormously the potential for examining specific early oncogenic events under a variety of circumstances. While the initial work w i l l focus on detailing the working parameters for employing the acrylamide cloning assay to detect newly transformed cells, later studies will be entirely devoted to exploiting the quantitative biological and molecular events involved in cell transformation by co-carcinogens. Wa felt that this new technique affords a unique tool for achieving the proposed objectives of describing the conditions which lead to radiation or chemically potentiated viral oncogenesis. The key observation that EA embryo cells (to the 10th day of gestation in the hamster) plate on acrylamidg gel surfaces with good efficiency while 11-14 day hamster embryo cells (EA adgitional biological parameter to explore. Since only EA EA tumor cells attach and grow on acrylamide, exploration of this parallelism should provide further insight into the significance of the similarities between fetal and tumor cells carrying re-expressed fetal do not adhere nor gr$w provides an fetal cells and
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The project conducted has been i n compliance with the contract as Project time distribution : Donadee Heffron, B.S., 20% time
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2.CoS g in, J.E. Jr. Enhanced Virus Trans for nation of Ea nster Embryo Capable of Inducing Transplantation Immunity Against SV4O Tumor Immunity i n Hamsters Immunized with Fetal Tissue.J.In i uno l., Tm or with Feral Antigens.In : Fetal and Eribrvonic ht i gens in Cancer I muniz ation with l!ouse Fetal An it c cns : Sup pre sive Ef ect on Cr otl rh of Leukemia - Virus Infected Cells and on Plasma Cell Tumors. I n : Embryonic and Fetal Antigens in Cancer, -I ,267 - 280, 1971. SV40 Oncogene s is with Human Fetal Antigen.Mature, -233:194-135, 1971, Sine con it ant Immunity toSV40 Tumors i n Embryoma-Bcnrins Ymstcrs. Dicrlan, P.,Anderson,N.G.,and Co gc in, 3.11. Jr.Iri muniz ation Against Tumors with Fetal Antigens : Detection 0': Immunity bythe Colony Inhibition Test and Adoptive Transfer. Embryonic and Fetal Antigens
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Refer cn ces, con t ' d with Ra usc her Virus and on Plasma Cell Tumors. Proc.tlat ' l Acad. Cyclic Ml? Levels inthe Developino, Hmster Fetus : A Correlation .with the Phas,ing of Fetal Anti ~ e ni n Eremhrpqe Maturation. Transformed Cells. Pe mbr an es and Viruses i n Immunopathology, Surface Auto antigens on Nembrancs of Fetus and Turnors. Adv. i n Influence of Parity Status o f Donor Females o n Imnunogenicit;: of Fetal Tissue and on Imnune Cell Cyto toxicity.hoc.Nat ' l . Acad. Prevention of Simian Virus 4.0 Tumors by Ka nster Fetal Tissue : Si ali c Acid to the Expression0 : Fetal Antigens Fn the Developing and Phase SD cci fic Fetal Antigens : An Evaluation of ? heir Role Cancer Cells.In : Developmental Aspects OE Carcinogen es is and mental Biology. (Ed.) Thomas King, Academic Press (;Jew York) N , (3. Proposed Ycchanisms by Nhich Autochclionous Tumors Escape Immune R e j e c t i o n . Cancer Res., 3 4 : 2 0 9 2 - 2 1 0 1 , 1 ? 7 4 .
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and Utility.In the Press Cancer Res.; June 1976. Transplantation Resistance Elicited by Fetal Tissues i n > lice. Huriioral Antibod:J Response and Tumor
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