(1) Joint seminar of “Molecular Mechanisms of Lymphoid Cell Activation and Application to Tumor Immunology”
This U.S.-Japan Cancer Research Cooperative Program Meeting was held in Makaha, Hawaii at the Sheraton Makaha, February 8-11, 1988. This format involved a combined meeting of two groups, one headed by Drs. Richard Hodes and Toshiyuki Hamaoka concerned with tumor immunology, the other group headed by Drs. Tadamitsu Kishimoto and Stanley Korsmeyer and focused on molecular immunology and molecular diagnosis. This meeting involved overall participation of 14 Japanese and 13 U.S. scientists, who met for four days to discuss recent finding in lymphoid cell activation with emphasis on molecular mechanisms.
I. The first session dealt with molecular events in lymphoid cell activation
Kathleen Kelly presented the results of studies which have characterized gene induction in human peripheral T cells following mitogen activation. Resting human peripheral blood T cells were activated with PHA and PMA in the presence of cyclohexamide for 4 hours. mRNA was isolated from such cells and cDNA was prepared. This cDNA was subtracted with mRNA from resting T cells in order to isolate cDNA specied which were expressed in a unique or quantitatively enhanced fashion in activated T cells. Cross-hybridization has revealed the presence of 57 unique genes to date. These have been analyzed to identify previously known genes. The majority of clones generated by this subtractive approach appeared to be novel. Recent studies have characterized families of these activation genes based upon the regulation of their expression in response to distinct signals, and in association with HTLV-I transformation. Further characterization of regulation and sequencing of these novel genes is in progress to determine their characteristics and role in lymphoid as well as in malignant cell activation. Roger Perlmutter then described his findings in a study of T cell lineage specific protein tyrosine kinase. The kinase lck is expressed only in lymphoid cells, including T cells. Regulation of expression of this lck gene during T cell activation has been characterized. In one experimental circumstance, two signals (PMA + ConA) are both needed for induction of lymphokines as well as for inhibition of lck gene transcription. The relationship of these kinases to oncogene function and cell transformation was discussed. Junji Yodoi discussed the increased expression of IL-2 receptor genes in HTLV-I transformed cells. The receptor gene is not rearranged, and the mechanism of transformation in these cells is unclear. A factor derived from ATL cells which is distinct from IL-2 is capable of inducing high affinity IL-2 receptors on cell lines. The ATL derived factor (ADF) has been purified, a monoclonal antibody to this product generated, and a cDNA clone isolated and expressed in cos cells, which should permit the further characterization of this factor. A monoclonal antibody termed YTA-1 has been produced against a T cell line, and this antibody has been demonstrated to accelerate the processing of IL-2 and IL-2 receptor. Further characterization of the determinant recognized by this antibody is in progress. Ko Okumura has accomplished the cDNA cloning of the mouse homolog of CD2. Both membrane and secreted forms have been expressed and a monoclonal antibody against the mouse CD2 has been generated. This has permitted the analysis of expression of CD2 during thymic ontogeny and studies are currently in progress to assess the subpopulation expression and functional role of the CD2 molecule. Kazuo Sugamura has analyzed growth signal transduction in IL-2 dependent T cells. He has demonstrated that the HTLV-I pX gene product p40tax activates transcription of IL-2 as well as IL-2 receptor!!
!and!!
!chains in the Jurkat cell line. Variant cell lines dependent upon either TPA or cholera toxin have been selected and it has been demonstrated that these lines differ in their susceptibility to growth inhibition by forskolin. This distinction implies that the two cell lines selected for differential growth requirements are differentially susceptible to cAMP inhibition. Kozo Kaibuchi discussed the existence of multiple pathways for protein kinase C (PKC) and GTP binding proteins in the signalling of growth factors. Differential susceptibility to inhibition by a series of pharmacologic agents has suggested the existence of different PKC activation pathways. At least 4 PKC genes have now been cloned and at least 2 types of activity can be distinguished in smooth muscle on the basis of susceptibility to inhibition. Toshiaki Katada discussed the novel function of the!!
!subunits of GTP binding proteins. The mechanism by which GTP binding proteins function in signal transduction was discussed. Stimulation of receptors by extracellular signals causes dissociation of coupled GTP binding proteins into their!!!and!!!subunits, together with release of GDP from the a subunit. Molecular mechanisms for the interaction of GTP binding protein subunits with effector enzymes such as adenylate cyclase were discussed. Kenji Sobue described the role of cytoskeleton interacting calmodulin binding proteins (cytocalbins) in regulation of the cytoskeleton. Shape, cytoplasmic movement and cell division are all influenced by Ca++ A series of cytocalbins appears to mediate the interaction of Ca++ concentration and cytoskeleton changes. Such events play potentially important roles in normal and transformed cell biology. Dr. Ken-ichi Yamamura of Kumamoto University initiated the discussions on the second day with a talk concerning transgenic mice as tools in the study of oncogenesis and autoimmune disease. His first transgenic experiment included an immunoglobulin heavy chain gene with the!!
!1 membrane segment. He noticed that B cells from such transgenic mice had a decreased stimulation following LPS as well as a one week delay in fetal expression of B cells. In a second very provocative set of experiments, Dr. Yamamura noted a marked host difference in the types of tumors generated by a c-myc construct bearing the immunoglobulin enhancer. In C57BL/6 mice tumors were noted within 4-7 weeks and were of B cell type located in the germinal centers. However, the same construct within C3H mice produced T cell origin tumors with a variable phenotype. Of considerable interest, was the fact that B6 x C3H F1 hybrids also produced T cell tumors. This suggests that there may be a dominant gene acting within C3H to produce a T cell phenotype. Finally, Dr. Yamamura reviewed his examination of the NOD, diabetic mouse model, in which there is a MHC as well as non-MHC locus contributing to insulinitis. NOD mice express no!!!-chain of the IE class-II antigen. Transgenic as well as F1 backcross studies reveal that the presence of an introduced IE halted the development of diabetes. This raises the possibility that the expression of IE eliminates an autoreactive T cell clone. II. Ig and Ig Receptors
Dr. Hitoshi Sakano of the University of California, Berkeley continued with a description of studies that dissected the molecular mechanism of immunoglobulin DNA rearrangements. Dr. Sakano had devised an informative system using retro-viral vectors in which both coding as well as signal joints could be rescued and analyzed. This work has revealed that the signal joints are precise and flush junctures of the two heptamer sequences while the coding joints are the sites at which significant exonuclease activity and N segment addition occurs. This model has been exploited to induce mutations in both the heptamer and nonamer motifs to examine the fidelity of the rates of recombination. Of the 7 bp heptamer, the last 3 basepairs of GTG adjacent to the endonucleotic cleavage are most crucial. The nonomers sequence appears to allow for more variation but once again possesses several rather invariant nucleotides. The length of the internal spacer region is more important in pre B cells which prefer 11 or 12 basepair versus a 13 basepair spacer while pre T cells are less stringent. Finally Dr. Sakano presented data on the purification of transbinding proteins that recognizes a heptamer motif. Of interest, the DNA binding activity purified in a 500 mM salt fraction while the endonucleolytic activity was most prominent in a 100 mM fraction. This may suggest that the immunoglobulin recombinase activity is composed of more than one protein.
Dr. Stan Korsmeyer of Washington University School of Medicine presented data on the deregulation of the Bcl-2-immunoglobulin fusion gene in follicular lymphoma and a polymerase chain reaction which is capable of amplifying that breakpoint. The normal Bcl-2 gene is expressed actively in pre B cell differentation but is quiet in mature resting B cells. Its transcription is induced by a variety of activation stimulants and B cell growth factors. The gene is down regulated with the induction of differentation. In contrast, translocated Bcl-2 genes in lymphoma cells have an inappropriately high level of expression for that mature stage of B cell development. The stability of the fusion message is unchanged indicating a marked deregulation of the transcription and processing of this gene. Transfection of Bcl-2 constructs prove that it functioned as a proto-oncogene converting B cells to tumors in nude mice. Finally, a sensitive polymerase chain amplification using thermophilic polymerase was capable of detecting one lymphoma cell bearing a single template copy of the translocation in a background of 106 normal cells. This approach markedly refines the detection of minimal residual disease and also provides an approach for detecting rare events such as interchromosomal translocations in recombination systems.
The final speaker in the section on immunoglobulin genes and immunoglobulin receptors was Professor Tadamitsu Kishimoto of Osaka University. He discussed their most recent information concerning the structure, function, and regulation of the CD23 molecule which serves as the FC receptor for IgE on B cells. A soluble form of the Fc!!
!Receptor was produced that allowed the definition of the affinity of this receptor for IgE. The expression of FceR proved to be B cell specific and missing from T cells. Epstein Barr Virus was noticed to increase the expression on B cell lines but was not related in Burkitt Lymphomas. The use of anti-CD3 antibodies also caused an increase in proliferation of normal B cells. The EBNA-2 molecule proved to be the identifiable trans-acting factor responsible for transcription of CD23 in B cells but not fibroblasts. Importantly, the physiologic stimuli of II-4 was shown to increase CD3 expression while!!!-interferon proved to inhibit it. III. The next session involved the T cell receptor and its structure and function
Dr. Richard Klausner of the NIH reviewed their extensive work characterizing the CD3 molecule in the mouse. Current data would indicate that CD3 may be as many as 7 chains. A number of cell mutants have been created which define the sites of interaction between these multiple chains. The most recent chains are zeta which appears to be the limiting chain for complex formation and is p-16 in molecular weight. The p22 associated molecule appears to have a unique peptide map and lacks carbohydrate and is referred to as the Eta chain. Two classes of CD3 molecules may exist in association with the T cell receptor. 80% appear to possess a zeta homodimer were as 20% Posses zeta-eta heterodimer. Whether these receptors define different cellular subpopulations or are each present on individual T cells is under active investigation. Both eta as well as zeta deficient variants were examined and suggested that eta may prove to be a developmentally regulated signal to activate T cells while zeta may have a predominant role in intracellular trafficking decisions.
Dr. Hideo Yamagishi of Kyoto University reviewed his data on the extra chromosomal excisional products of T cell receptor gene joining. Dr. Yamagishi first noticed the presence of circular molecules by electron-microscopy in normal thymus but not long term clonal T cell lines. These proved to be excisional products of T cell receptor gene joining with junctures between the heptamer signals. Of interest, most of his excisional products that follow the V!!
!/J!!! recombination no longer have the!!
!T cell receptor present internally. This would be compatible with a distinct mechanism to delete the!!!locus. Of interest, Dr. Yamagishi has also noted the presence of circular extra-chromosomal molecules in non-lymphoid cells including HeLa which may imply the presence of other non-immunoglobulin related rearranging genes and perhaps provide a handle to isolate human autonomous replicating sequences and centromeres. Dr. Michael Brenner of Harvard University reviewed their most recent information on the!
!!T cells. There appear to be several different subpopulations of!!!T cells based on the utilization of the associated!!!chain. The differences are focused around a 48 basepair middle exon, referred to as CII. This may be present in the 1, 2 or 3 copies. C!!!2 possesses 2 copies and has a 40 Kd chain were as C!!!1 may use one copy or 3 copies. This accounts for the size heterogeneity noticed in these T cell receptors, yet all use the same!!!chain. Interestingly, all three forms of the!!!chain can be successfully expressed within the same T cell line. They noted one predominate variable region, V!!!1 being used in all of their!!!cDNAs. cDNAs were most compatible with two internal diversity segments. Considerable diversity does appear to be generated by N segment addition in this system. Dr. Jeffrey Bluestone of the University of Chicago followed with a discussion of their physiological data concerning the expression of the!
!!T cell receptor and its function within cytoxic T cells. They noticed that CD3 positive but CD4 negative, CD8 negative cells when activated would develop CD8. They were able to isolate CD3!!!cells that displayed alloreactivity to independent class I antigens. Of interest, both of these clones utilize C!!!4. There is accumulating evidence that there are preferential 8 associations with certain variable!!!regions being preferentially seen in the!!!T cells. Similarly, data is emerging that may indicate a differential use of the two joining segments of the 8 T cell receptor in thymic cells versus cells in the periphery. IV. The first session on Wednesday morning February 10 was devoted to Lymphokines and Lymphokine receptors
Dr. Toshimitsu Uede of Sapporo Medical College initiated this discussion with a presentation of his studies concerning the affinity conversion of human ll-2 receptors. A chimeric cDNA in which the extra cellular receptor was that of the Tac P55 molecule where as the transmembrane and intracytoplasmic portions were of the human insulin!!
!chain, was constructed. This was introduced into the mouse E1-4 cell line which had previously been shown to convert to high affinity II-2 receptors with the transfection of complete Tac. Provocatively, preliminary data suggested that the associated p75 molecule of the mouse did not require the transmembrane and intracellular aspects of Tac antigen. Instead the extracellular portions of Tac may be sufficient to create the high affinity complex with P75. Dr. Warner Greene of Duke University presented their data concerning the separate chains of the II-2 receptor, P55!!
!, P70!!! and the combined!!!high affinity receptor. The extremely short time association and disassociation constants of the!!!chain are in contrast with the long times of association and disassociation of the!!!chain. The final hybrid!!!high affinity receptor manifests a 37 seconds association constant but a very slow 285 minute disassociation. This has important implications for the selection of T cells for expansion in the presence of limiting concentrations of interleukin-2. In a second line of investigation, the Il-2 receptors promoter region was dissected with the identification of two internal direct repeats. These contain consensus binding sites that recognize an 86 Kd nuclear protein. This consensus motif would cross compete with the HIV direct repeat that serves as an enhancer for that virus. This provides an intriguing insight into the relationship of HIV replication and toxicity within dividing T cells. Dr. Tadatsugu Taniguchi of Osaka University followed with a description of their analysis of the interferon!!
!and!!!genes. This is a very complex promoter that contains both a negative regulatory element, a constitutive region as well as inducible elements. The inducible motif of AAGTGA functions as a virus inducible enhancer. A cell type specificity for this 30-40 Kd nuclear binding factor was also noted. Provocatively, modifications of already bound transbinding proteins were noted suggesting post-translational regulation. Dr. Kiyoshi Takatsu of Kumamoto University summarized their work on IL-5 also known as T cell replacing factor. The cloning of this gene enabled the production of abundant recombinant interleukin-5 and has resulted in the elegant description of its physiologic effects. An antigen primed B cell system reveals that IgA bearing B cells will respond to IL-5 as a maturational or help factor. Moreover, IL-5 will induce the growth of very early pro-B lymphocytes as well as serving as a differentiation factor in later development. Two populations of IL-5 receptors exist with a high affinity receptor at approximately 400/cell and a low affinity receptor of 7,500/cell. Crosslinking experiments reveal that the IL-5 receptor has a net molecular weight of approximately 45 Kd.
The final speaker in the lymphokine section of the meeting was Dr. Toshio Hirano of Osaka University. He reviewed their very extensive information on Interleukin-6 and its receptor. IL-6 has wide ranging effects well beyond its B cell differentiation role. It can serve as a nerve growth factor, hepatocyte stimulating factor, and inducer of CTL differentiation, as well as a growth factor for hybridoma and plasmacytomas. Provocatively, many multiple myeloma cells were shown to make IL-6 in addition to having interleukin-6 receptors on their surface. This raises the intriguing possibility that an autocrine growth cycle might exist within such maligancies. IL-6 receptors exist in both high and low affinity forms with the Kd of the high affinity being 1.7 x 10-11M with approximately 4,100 receptors per cell while the low affinity receptor is 10-9M at 24,000 receptors per cell. A candidate gene for the receptor was cloned from YT cells using the CDM8 expression vector. This 468 amino acid molecule confers low affinity IL-6 binding activity on fibroblasts. The molecule has a classic signal peptide binding domain and transmembrane and intracellular domains. It has no obvious homology with other receptors and has no associated kinase activity.
V. The fifth session of this meeting dealt with molecular mechanism of T helper cell activation and function
Toshiuki Hamaoka first described la restricted B cell-B cell interactions and their relationship to the B cell repertoire. T cell replacing factor II (TRF-II) is capable of inducing the polyclonal induction of B cell antibody responses. Studies employing anti-Ia antibodies and radiation bone marrow chimeras have demonstrated that the polyclonal response to TRF-II requires interactions between B cells, and that these interactions involve a recognition of “self” Ia determinants. The la recognition repertoire of B cells is determined during their maturation by the la type of bone marrow derived cells, not of radiation resistant host determinants as has been reported for T cell differentiation. Immune response gene-like phenomena have also been reported in the content of these B cell-B cell MHC restriction requirements. Charles Janeway discussed the specificity and function of CD4+ T cells. Two subpopulations of these cells, termed TH1 and TH2 have been characterized on the basis of differential patterns of lymphokine secretion. Dr. Janeway has described the ability of TH2 cells to function as specific helper cells for B cell responses, in contrast to the ability of TH1 populations to act as cytotoxic cells and as mediators of inflammation. The role of the CD4 molecule was analyzed by blocking and modulation experiments. It was concluded that at least three functional roles of these molecules exist: l) as an off signal; 2) in adhesion strengthening; 3) by interaction with the T cell receptor and the target cell ligand as a mediator of high potency T cell activation. A new monoclonal antibody, termed YCD45R, has been generated which appears to distinguish the TH1 and TH2 populations in the mouse and which will be applied to further characterization of the distinction between these subsets. Richard Hodes described two potential mechanisms contributing to specific T helper cell effector function in the activation of B cell responses. It was demonstrated that TH2 helper cells contain cytoplasmic granules and that granule exocytosis can be induced by stimuli delivered through the T cell receptor, but not through the IL-2 receptor. Such rapid release of pre-formed mediators provides a potential mechanism for the rapid and directed delivery of signals from the T helper cell to target B cells. Additional studies have demonstrated the presence in cell free supernatants from activated T helper cells of specific helper factor. Such factor is both antigen specific and MHC restricted in its capacity to promote otherwise T cell dependent responses. Monoclonal antibodies toward variable region T cell receptor structures have been employed to affinity purify the active factor in the supernatants, suggesting that they are highly analogous to or identical to the cell surface T cell receptor structure. David Perkins analyzed the molecular mechanism of antigen presentation in T cell recognition. A series of peptides derived from malaria organisms or from viral nucleoproteins were analyzed for their abilities to induce T cell proliferative responses. By using individual and joint peptides it was possible to detect the apparent stabilizing influences of one peptide on adjacent sequence as well as the creation of new hybrid determinents. The possible existence of conserved residues in a series of peptides recognized in association with the same la molecule was described, suggesting conserved motifs in the peptides recognized by T cells. Ronald Schwartz described a system of induced non-responsiveness in THI cloned cells. Antigen presenting cells (APC) which have been altered by fixation were unable to induce antigen specific proliferation in T cell clones. Nevertheless, exposure of these clones to antigen in the context of such treated cells or in association with synthetic lipid bilayers resulted in the specific unresponsiveness of those clones to subsequent challenge with antigen. The mechanism of this unresponsiveness was assessed at a molecular level. Tentative conclusions to date are that the presentation of antigen by defective stimulus results in an increase of intracellular calcium in the absence of required costimuli. The consequence of this event is the subsequent unresponsiveness of cells to further challenge through the T cell receptor.
VI. The sixth and final session of the meeting dealt with T cell function and anti-tumor immune responses
Toshiuki Hamaoka described antigen recognition mechanisms of antitumor T cell subsets involved in host tumor resistance. The roles of both CD4+ and CD8+ tumor immune cells in antitumor responses were characterized. This was accomplished in a system employing in vivo implanted chambers which allowed the analysis of induction of nonspecific bystander mechanisms of tumor cells lysis. It was demonstrated that both CD4+ and CD8+ T cells can produce antitumor effects. Antigen presenting cell requirements were demonstrated at the level of antigen recognition and T cell activation. The role of defined lymphokines in mediating these effects was discussed. Philip Greenberg further described the role of T effector cells in host antitumor responses. In a syngeneic mouse leukemia model, it was demonstrated that a combination of cytoxan and immune T cells was required to establish long term cures. The long term presence of persisting antitumor T cells was shown to be necessary for this effect. Cloned tumor specific T cells were generated and roles for endogenous IL-2 and IL-4 were evaluated in proliferative T cell responses to syngeneic tumors. The nature of tumor antigens recogni7.ed by T cells is currently being studied through the use of transfectants with cloned viral genes.
(2) Seminar on “Molecular Biology of Cancer Cells”
The U.S. Japan seminar on the molecular biology of the cancer cell was held in Kyoto, Japan on April 4-6, 1988. The meeting began with introductory comments by the host, Dr. Shigetada Nakanishi, who outlined the program. The first session began with a presentation by Dr. Watanabe on lymphocyte differentiation in gene expression control. Dr. Watanabe described studies from his laboratory investigating how genes involved in lymphocyte expression and differentiation were controlled. Dr. Watanabe’s laboratory has purified a transcription factor from B cell nuclear extracts which is essential for expression of immunoglobulin heavy chains.
Dr. B deCrombrugghe described his studies on regulation of collagen expression by the growth factor TGF-!!
!. There is an upstream TTGGCA sequence (NF-1 site) which is essential for the effect of TGF-!!!. In some constructions, addition of a DNA fragment containing this sequence confers TGF-!!!responsiveness on gene expression. Next, Dr. Ron Evans from the Salk Institute discussed his studies on gene regulation by steroid and thyroid hormone receptors. Dr. Evans described the novel and powerful technique he developed to study regulation by receptors. By transfecting two plasmids into a cell, one encoding a receptor and one encoding a receptor responsive element. Dr. Evans developed a new method of assaying receptor function. This method was then used to determine the function of various portions of the receptor genes he cloned. Dr. Evans also used his initial isolate of one receptor to isolate a large gene family of receptors. This family includes receptors for thyroid hormone, receptors for various steroid hormones, receptors for vitamin A and other related receptors. All the receptors have a common DNA binding element but different receptor binding regions. By employing this novel gene isolation method, Dr. Evans has developed an approach that will enable many other scientists to isolate a variety of DNA regulatory proteins. While the DNA binding region of all these proteins is similar, the mechanism by which the DNA-protein interaction is controlled differs among the different proteins.
Dr. Tadashi Yamamoto then described his laboratory studies on the transforming potential of protooncogenes of the tyrosine kinase family. Dr. Yamamoto described the isolation of several protooncogenes from this family and discussed studies on their comparative sequences. Recently, Dr. Yamamoto’s laboratory has begun to investigate expression of these protooncogenes in normal tissue to determine the role these important proteins have in differentiation. Dr. H. Ariga then described his recent studies on the regulation of DNA replication and RNA transcription by the c-myc protein. Dr. Ariga has detected effects of c-myc protein on DNA replication which represents a unique action of this protein. Dr. Ariga described the basis of his assays for c-myc protein function. Following this Dr. Ariga described how c-myc regulates transcription. The meeting was then adjourned for the day.
The next morning Dr. Sankar Adhya described his studies on elements that produce positive and negative control of gene transcription. Dr. Adhya began his presentation with a description of an early paper in which Dr. Nakanishi, chairman of this symposium, had described effects of the galactose repressor protein gal operon transcription. Dr. Adhya then summarized the history of his studies on the regulation of gal transcription and next described in detail current models for how gal transcription is regulated. Dr. Adhya described his studies on the identification of two operator sites for gal transcription control and the need for both operator sites to be occupied to achieve maximal repression. Next, Dr. Adhya described how two molecules binding at distant sites on DNA work together to control transcription. All the data supports a model in which the DNA forms a loop structure dependent on binding of regulatory proteins at two distant sites. These results have clear implications for the mechanism by which regulatory proteins control gene expression in eukaryotic cells. In eukaryotic genes, binding sites for many different regulatory proteins are often found to occur.
Dr. T. Honjo of Kyoto University described a series of studies on the regulation of growth and differentiation of lymphocytes by lymphokines. He summarized his classic work on the isolation of several lymphokines, and then he described recent experiments on factors that control the differentiation of B cells which indicate that a novel molecule, perhaps a new lymphokine, is involved in lymphocyte differentiation. Dr. Honjo described how a system is developed in order to study lymphokine control.
Continuing in the theme of the biology of the cancer cell, Dr. M. Takeichi described the structure and function of cadherins. These are molecules located in the plasma membrane that are responsible for calcium dependent binding of cells to each other. Cadherins have an important role in normal tissue architecture and in differentiation and development. Recently, Dr. Takeichi has begun to examine the possible role of cadherins in tumor metastasis. Failure of cells to synthesize cadherins should lead to decreased tissue integrity and could lead to the invasion and spread of tumor cells.
Dr. H. Imura discussed studies done in his laboratory on glucose transport mechanisms. He reviewed the structure of glucose transport proteins and discussed studies done in his own laboratory on the mechanism by which glucose is transported. The structure and regulation of the genes encoding two glucose transporters (a placenta/brain transporter and a liver/kidney transporter/were discussed.
The next session concerned the role of receptors and second messengers in the regulation of cell behavior and cell growth. Dr. Mark Caron from Duke University described studies on the structure and function of catecholamine receptors. Dr. Caron described how he used affinity chromatography to isolate sufficient amounts of the!!
!-adrenergic receptor to obtain structural information. Based on this information, he and his colleagues were able to isolate a cDNA clone for a catecholamine receptor. The sequence of this clone indicated the receptor was very similar in its structure to a protein involved in vision termed rhodopsin. This protein receives a light signal and converts that signal into a biochemical intracellular signal involved in the process of vision. Following the isolation of the initial catecholamine receptor, it was possible to isolate other members of the receptor family by screening selected cDNA libraries. Mutational studies on the receptor have indicated which regions of the protein are probably required for hormone binding, for interaction with G proteins and for other functions. Next Dr. Shigetada Nakanishi of Kyoto University described a new method of cloning receptors for hormones and growth factors. Dr. Nakanishi has developed an extremely sensitive method for receptor gene isolation which should enable investigators to isolate many receptors that are present in extremely small amounts in cells. This method involves the production of full length cDNA’s, transcription of the cDNA into RNA and microinjection of RNA into oocytes. If the receptor is coupled to a change in membrane function such as an increase in chloride transport, it is possible to detect extremely small amounts of the protein produced by the injected RNA. The electrophysiological measurements which reveal the influx of chloride are extremely sensitive and single membrane events can be detected.
Dr. M. Ui described his studies using pertussis toxin to activate adenylate cyclase. Pertussis toxin works through the G protein system to control adenylate cyclase activity. Using pertussis toxin as a tool, he was able to clarify how G proteins are involved in regulation of adenylate cyclase activity.
Dr. I Uno has used a genetic approach to investigate second messenger systems including phosphoinositol and the PIP2 cascade in yeast. The advantage of studying yeast is that the cells have very similar metabolic properties to higher eukaryotic cells but because of their simple genome and rapid growth, it is possible to isolate many mutants in yeast metabolic control pathways. Dr. Uno has been able to isolate mutants throughout the PIP2 cascade and clarify the sequence of metabolic products produced in this system.
The last session was devoted to the use of recent advances in molecular biology and cell biology to devise new types of cancer treatment.
Dr. I Pastan described his studies on the creation of new types of cytotoxic agents. Dr. Pastan first described his studies on the EGF receptor and provided evidence that the EGF receptor can function as an oncogene when overexpressed in normal cells. Overexpression is achieved by placing a full length cDNA for the EGF receptor in a retroviral expression vector. Cells infected with the EGF receptor virus show transformed characteristics in tissue culture. Cells that also overproduce the EGF receptor form tumors in nude mice. He then described how pseudomonas exotoxin (PE) can be directed to EGF receptor bearing cells. To do this, the cell recognition portion of PE is deleted and replaced with the growth factor transforming growth factor alpha (TGF!!
!) to create a chimeric molecule termed TGF!!!-PE40. These molecules bind to, enter and kill cells bearing EGF receptors. Dr. T. Tsuruo of the Cancer Institute (Tokyo) has made a monoclonal antibody to a 170,000 Mr protein found on the surface of multidrug resistant cancer cells. He has used his antibody (MRD16) to study drug resistance. Dr. Tsuruo showed that drug resistant cells have an increased sensitivity to drugs after exposure to MRK 16 suggesting its possible therapeutic use. He also has used the antibody to isolate P170 and showed that the isolated protein has ATPase activity. Membrane vesicles from drug resistant cells were found to bind (3H)-vinblastine and the binding was enhanced by ADP sugesting interaction of vinblastine and ATP with the same protein.
Dr. M.M. Gottesman concluded the meeting by presenting an overview of the ways in which cancer cells avoid being killed by cytotoxic chemotherapeutic drugs. His presentation emphasized the expression of the multidrug transporter (P170) in multidrug-resistant cells. The MDR1 gene encoding this transporter has been cloned and sequenced. It encodes a plasma membrane protein which uses the energy of ATP to pump cytotoxic drugs out of cells. This pump is normally expressed in kidney, liver, small and large intestine and adrenal gland, and may be involved in excretion of cytotoxic products in the diet and toxic endogenous metabolites. Preliminary clinical studies indicate that it is expressed by many tumors, such as adenocarcinomas of the kidney, liver, colon and adrenal, which are intrinsically resistant to chemotherapy, and that it may be expressed after therapy in many other tumors which are initially sensitive to chemotherapy, but become resistant during the course of therapy.
It was the general feeling of the participants that enormous progress has been made in understanding the molecular biology of the caner cell. This U.S.-Japan Cooperative Cancer Research Seminar highlighted many important recent discoveries. Cooperation between U.S. and Japanese scientists has helped speed research on the basic biology of human cancer.
SEMINAR AGENDA AND PARTICIPANTS
(1) SEMINAR ON MOLECULAR MECHANISMS OF LYMPHOID CELL ACTIVATION AND APPLICATIONS TO TUMOR IMMUNOLOGY
Honolulu, Hawaii, February 8-11, 1988
AGENDA