(1) Seminar on “Antigen presentation and immune cell interaction”
This meeting, organized by Drs. Hiromi Fujiwara and Richard Hodes, was held in Osaka, Japan. There were 11 Japanese and 6 U.S. participants in a meeting organized to discuss the roles of antigen presentation, cell-cell adhesion, and signal transduction during immune cell activation.
I. Antigen Presentation
The first session of this meeting dealt with the process of antigen presentation during immune response. Dr. Shun-Ichi Kumagai presented the results of recent studies suggesting the involvement of the self peptides in the recognition of self la by autoreactive HLA-DR and HIA-DQ specific human T cell clones. Mouse L cells transfected with human DR or DQ were inefficient targets for autoreactive T cells unless these L cells had been previously exposed to lysis of autologous or allogeneic B cells. These findings suggest that autoreactive T cells, including those involved in autoimmune disease, may recognize self antigens in association with self MHC class II molecules. Dr Toshiyuki Hamaoka presented data characterizing the antigen presenting cells present in tumor-bearing mice in vivo. APC harvested from tumor-bearing animals but not from normal animals, were capable of inducing potent tumor resistance in vivo. This resistance was shown to be T cell mediated and tumor-specific. It thus appears that systemic antigen presenting cells in tumor-bearing animals are functionally able to present this antigen and to prime a normal host for anti-tumor immunity. Dr. Toshiaki Mizuochi described a defect in antigen presenting cell function induced by murine retrovirus infection that serves as a mouse model for AIDS. In both proliferative responses and cytotoxic T cells generation in vitro, APC from virus infected animals had progressive defects in antigen presenting capacity from one to five weeks after viral exposure. This APC defect was not overcome by the addition of cytokines, and may represent an important part of the immune suppression observed in AIDS-like retroviral infections. The cell biology of antigen presentation was discussed by Dr. Frances Brodsky, who described studies indicating the complex pathways of intracellular processing that are available for presentation of both endogenous and exogenous antigens in association with class I and class II MHC products. The combined use of functional and electron microscopic methodologies in these studies allowed an appreciation of alternative pathways for antigen processing and association with MHC molecules.
II. Cell-Cell Adhesion
A number of presentations discussed the role of cell-cell adhesion in lymphopoiesis. Dr. Kensuke Miyake discussed the role of lymphoid cell adhesion to stromal cells in lympho-hemopoiesis. Results were first presented demonstrating the role of CD44 in both B cell and granulocyte development. In addition, results of more recent studies were presented which indicate the role of a VLA4-ligand interaction that is of particular importance in B cell lymphopoiesis. Dr. Toshimitsu Uede described a new adhesion molecule involved in thymocyte-thymic epithelial cell interaction. This new antigen, 7D3 is involved in a pathway distinct from the known LFA-1/ICAM-1 and CD2/LFA-3 pathways. The 7D3 antigen, which is expressed on thymocytes, bone marrow cells, fetal liver cells and thymic epithelium is involved in the binding of thymocytes to thymic epithelial cells and represents a third and novel pathway for this interaction. Dr. Hiromi Fujiwara also discussed the adherence of immature thymocytes to thymic stromal cells. In this case, adherence to fibronectin was shown to be critical to both growth and differentiation of thymocytes. Blocking experiments suggest that two different sites on the fibronectin molecule are involved in this adherence. Potential roles of integrins such as VLA4 or VLA5 and their regulated expression on immature thymocytes were discussed.
In addition to their role in lymphoid cell development, a role of cell adhesion molecules was also demonstrated in mature lymphocyte activation in several systems. Dr. Edward Clark discussed several aspects of the regulation of B cell adhesion to T cells. Data were first presented demonstrating the role of tyrosine phosphorylation as well as inositol phospholipid turnover and a rise in intracellular calcium in signalling the B cell through surface lg. In addition, a role of CD45 mediated dephosphorylation in B cell activation was shown. In studies of T cell dependent B cell activation, the role of interaction between CD28 and its recently defined ligand B7, as well as interaction between LFA-1 and its ligand ICAM-1, was shown. Results suggested a sequential cross talk between T cells and B cells mediating the interaction of both of these cell types during T-B interaction. Dr Richard Hodes then discussed the role of CD44 in B cell activation and adhesion. As a result of specific B cell activation by IL5, a unique population of CD44-bright, CD45-dull, la-dull cells was induced. Cell fractionation studies demonstrated that this population mediated the proliferative and Ig secretory activity of the activated B cell population. In addition, it was shown that CD44 expressed on this subpopulation of cells mediated binding to the ligand hyaluronate. These findings suggested a functional role for CD44 in mediating the trafficking and adhesion of activated B cells to extracellular ligands in vivo. Dr. Nobuo Sakaguchi summarized the construction of a B cell specific cDNA library by subtractive hybridization and the isolation of one clone, mb-1, which encodes a IgM-associated protein on the surface of B cells. The association of this protein with the Ig receptor resembles the association of CD3 structures with the T cell receptor complex. The functional role of this gene product is currently under study. In the final presentation of this session Dr. Hideo Yagita described the identification of a novel adhesion molecule involved in cytotoxic cell-target cell interactions in a murine system. A monoclonal antibody, RMV-7 shown to inhibit CD2/LFA-1-independent LAK cells cytotoxicity and binding to target cells. The antigen identified by this antibody is expressed predominantly on myeloid cells in vivo but appeared on peripheral T cells late after mitogenic stimulation. The RMV-7 antigen is a heterodimer composed of a 140kDa chain and a 95kDa chain, similar to the integrin family of molecules. RMV-7 blocked LAK cell binding to fibronectin, fibrinogen and vitronectin, but not to laminin or type IV collagen, indicating that the RMV-7 defined molecule is a unique extracellular matrix receptor. The cytotoxic activity of LAK cell well as CTL was inhibited by the RMV-7 as well as anti-fibronectin antibodies, indicating the importance of this receptor-ligand interaction in target cell binding and cytotoxicity. Immobilized antibody RMV-7 was also able to synergize with other stimuli in the activation of T cells, indicating a role for this receptor in signalling.
III. Signal Transduction
This session discussed the role of a number of cell surface receptors in transducing positive and negative signals for lymphoid cell activation. Dr. Takashi Saito discussed the role of CD2 in T cell activation. It was first demonstrated that CD2-mediated activation could occur in cells which did not express the T cell receptor/CD3 complex, but that such responses were observed only when cells expressed high levels of CD2. The concurrent expression of TCR/CD3 lowered the level of CD2 required for activation via the latter pathway. The expression of CD2 also enhanced the response of T cells to antigen-specific activation when B cells were utilized as APC. Thus, CD2 has important roles in T cell responses as both an activation molecule and an adhesion molecule. Dr. Matthew Mescher analyzed the role of CD8 in T cell responses. It was shown that purified class I protein is the ligand for CD8-specific adhesion. However, CD8-dependent adhesion occurs on T cells which have been activated through the TCR. This activation does not involve a detectable change in cell surface density of CD8, but nevertheless alters the effective affinity of CD8+ cells for class I ligand. The T cell signalling required for this activation of CD8 does not require detectable alterations in calcium concentration or hydrolysis of PI. These results thus represent an intriguing example of receptor-mediated adhesion which is regulated by cell activation through another surface receptor. Dr. John Imboden described differential aspects of signal transduction in T cells and NK cells. NK cells, which do not express a TCR, can be induced by interaction with their specific targets to undergo PI hydrolysis. This response can be modulated by perturbation of CD2. A monoclonal antibody generated to NK cells detected a new cell surface molecule (gp35) which is also expressed on a subpopulation of rat thymocytes and on all mature leukocytes. Anti-gp35 antibodies are capable of activating both NK cells and T cells. gp35 appears to be physically associated with CD2 on the cell surface and may regulate the coupling of CD2 to signalling pathways. Dr. Carl June reviewed data indicating a critical role of tyrosine phosphorylation in T cell activation. Triggering of T cells through CD3 or CD2 was shown to induce different patterns of tyrosine substrate phosphorylation. Antibodies to another cell surface molecule, CD28, on T cells were capable of synergizing with PKC stimuli to produce T cell activation. Two additional presentations dealt with the role of cellular oncogenes in normal cell function. Dr. Shin-Ichi Nishikawa described the role of c-kit in embryogenesis and hemopoiesis. A series of elegant experiments demonstrated the function of c-kit in melanocyte migration during fetal development. Anti-c-kit antibodies were also able in vivo to inhibit generation of myeloid and erythroid cells, while in contrast B cell lymphopoiesis continued normally. Analysis of colony forming precursor cells indicated that c-kit function is involved in the self renewal of the most immature colony forming precursors. Finally, Dr. Tadashi Yamamoto analyzed the possible involvement of src-like protein tyrosine kinases in lymphocyte signalling. Gene expression of several of the src family members was analyzed in lymphoid cells. It was found the fyn and lyn genes are preferentially expressed in B and T lymphocytes respectively in peripheral lymphocytes. The fyn gene was over expressed in abnormal T cells characterizing certain autoimmune diseases. It was also demonstrated that the lyn protein is associated with membrane bound Ig in B cells. Thus, the fyn and lyn proteins are suggested to be important in lymphocyte signalling by the antigen receptors on T cells and B cells.
The final session of the meeting involved a discussion which attempted to integrate patterns of expression of cell adhesion receptors with potential roles in cell function. Sets of cell adhesion molecules were described which were either 1) expressed only after cell activation, or 2) showed increased levels of expression after cell activation, or 3) showed decreased levels of expression after activation. An enormous complexity exists in the potential for signalling through multiple cell receptors which in turn influence the expression and functional status of additional cell surface molecules. Together, these interactions undoubtedly serve to modulate in vivo activation and trafficking of cells including those of the immune system. The presentations made at this meeting, as well as the extremely interactive discussion which followed, provided a review of the most recent findings in the area and an opportunity to share perspectives on the interpretation of these new findings.
(2) Seminar on "Signal Transduction and Gene Regulation in Cancer”
This seminar was held on October 10-12, 1990 in Building 31, Conference Room eight at the NIH m Bethesda, MD. The organizers were Dr. Glenn Merlino, National Cancer Institute, Bethesda, Maryland and Dr. Shunsuke Ishii, RIKEN Tsukuba Life Science Center, Tsukuba, Ibaraki. There were eight participants from the United States one from Canada, and seven from Japan. The purpose of the seminar was to discuss and exchange information on a wide range of cancer-related topics including oncogenes, growth factors and signal transduction, gene regulation and trans-acting factors, and transgenic mouse models. An additional primary goal was to encourage and establish future cooperative interactions between researchers in the United States and Japan.
Ras and Signal Transduction
Dr. Yoji Ikawa discussed recent results on the Krev-1 gene and the related c-ras genes. Krev-1 encodes a ras-related protein which can induce flat revertants when introduced into v-Ki-ras-transformed mouse NIH3T3 cells. He constructed a series of mutations in Krev-1 cDNA and tested their biological activities in a number of transformed cell types. Substitutions in Krev-1 of the amino acid residues within the putative guanine nucleotide-binding regions (Asp17 and Asn116) the putative effector-binding domain (residue 38), the putatitve acylation site (Cys181), and at a unique Thr61 residue all decreased transformation suppressor activity. In contrast, other point mutations (Gly12 to Val12 and Gln63 to Glu63) significantly increased transformation suppression. He suggested that in response to negative growth-regulatory signals, the Krev-1 gene product was regulated by GDP/GTP exchange, like other G-proteins. Interestingly, when chimeric genes were created consisting of fragments of c-Ha-ras and Krev-1, the determinant for transformation suppressor activity was found to reside within the N-terminal third of Krev-1 where the putative effector-binding domain and surrounding divergent amino acids are found. The other regions are almost interchangeable. He closed with a discussion of future assays which could be used to further elucidate the function of this important protein.
Dr. Douglas Lowy continued on the theme of transformation suppression. He elaborated on the structural relationship between members of this family of proteins. He also described the creation and use of v-Ha-ras/Krev-1 chimeras in transformation suppression assays. He discovered that the divergent amino acids surrounding residues 32-44 determined whether the chimeric protein would induce transformation or suppression; suppression of ras transformation represented an effector function of Krev-1; and Krev-1 induced suppression was probably due to interference with the ability of ras to stimulate its target. Dr. Lowy also described studies on rasGAP, which interacts with ras and acts as a negative regulator. Transfection of rasGAP expression vector into transformed cells or co-transfection of rasGAP and activated ras into normal cells resulted in inhibition of ras transformation. He also discussed the effects of lovatstatin on ras-induced transformation. Lovatstatin inhibits cellular growth but is independent of ras transformation.
Dr. Hiroaki Ohkubo introduced the mammalian tachykinin system. Three peptides were described: substance P, substance K and neuromedin K. These share common biological activities including sensory transmission in the nervous system and contraction and relaxation of peripheral smooth muscles. Dr. Ohkubo described success at cloning functional cDNAs encoding receptors for each of these three peptides using an electrophysiological Xenopus oocyte assay. The three receptors share gross structural features, and regions of high amino acid conservation. Despite these structural similarities, they maintain selectivity for binding specific peptides. His results provide great insight into the molecular basis of the functional diversity of multiple peptide receptors.
Dr. Tadashi Yamamoto then presented studies on two members of the src protein-tyrosine kinase family: fyn and lyn. Members of the src family possess a powerful tyrosine kinase that resides in the internal portion of the plasma membrane, but lack extracellular and hydrophobic transmembrane regions. fyn and lyn are selectively expressed in T and B lymphocytes, respectively. Overexpression of fyn in specific T cells results in the development of a lymphoproliferative autoimmune disease in specific mutant mice (i.e., lpr). Further study revealed that fyn is probably physically associated with the TCRCD3 complex. A similar story was described for lyn, which was shown to be associated with B cell membrane-bound IgM. He concluded with the hypothesis that products of the fyn and lyn genes may be crucial for antigen receptor-specific lymphocyte signalling.
Mouse Models for Oncogene Action
Dr. Paul Overbeek began this session by describing his recent work on targeted overexpression of growth factors and oncogenes in the eye. Transgenic mice that expressed either epidermal growth factor or transforming growth factor!
!!in the lens of the eye exhibited an altered interaction between the embryonic lens and cornea, resulting in dysmorphology. He postulated that these factors could serve as inappropriate morphogenetic signals that interfer with normal differentiation patterns. He also discussed transgenic mice that expressed the abl oncogene in the lens of the eye. In this case abl caused a dysplastic transdifferentiation of the lens epithelial cells into fibroblastic cells. Dr. Overbeek concluded that the use of transgenic mouse models for eye development will continue to be informative in the future. Dr. Robert Hammer also used transgenic mice to create models for urogenital disease Mice were generated bearing the human papilloma virus 18 promoter driving expression of the open reading frame of E6-E7. Males from lines of these mice showed enlarged seminal vesicles and preputial glands, and females from similar lines exhibited cervial fibrosarcomas. He concluded that the E6-E7 region of HPV-18 can induce hyperplasias and neoplasias in specific tissue types of transgenic mice. The tissue specificity probably reflects transcriptional selection by the HPV-18 promoter. Furthermore, these mice may be valuable for analysis of E6-E7 function, and for the identification of cellular targets of these viral gene products.
Dr. Motoya Katsuki developed transgenic mice carrying human c-Ha-ras genes driven by their own promoters. Half of the mice from these lines developed tissue-specific tumors within 18 months. Most of these were angiosarcomas. All angiosarcomas were found to contain point mutations in the ras transgene at the 61st codon from Gln to Leu, but not in the endogenous murine ras genes. Other tumors were described, such as skin papillomas and Harderian gland adenocarcinomas. Treatment with methylnitrosourea resulted in the further development of forestomach papillomas possessing point mutations in the 12th codon from Gly to Asp or Ser, but again only in the ras transgene. He concluded that specific somatic mutations of human c-Ha-ras genes were closely associated with development of tissue-specific tumors.
Dr. Janet Rossant concluded this session with a clear and detailed presentation of the use of targeted homologous recombination and embryonic stem cells to produce null mutations of specific genes in the mouse germ line. This technology provides the best approach to achieve total inactivation of genes of interest. She then described the production of a null mutation in the mouse N-myc proto-oncogene in the mouse germ line. This gene was particularly interesting since it is associated with transformation and has been detected in the mesoderm migrating through the primitive streak and later just prior to terminal differentiation. Inactivation of both N-myc alleles does not prevent fetal development, but does result in newborn lethalities. Dr. Rossant finished by presenting a convincing message that this was a powerful approach whose time had come.
Gene Regulation
Dr. Benoit deCrombrugghe began this session by presenting recent findings on the regulation of the type I collagen genes. He discussed transgenic mice harboring from 350 to 2000 bp of 5' flanking region of the a2(I) collagen gene. Both constructs directed expression of a marker gene in tendons, bone and skin. He is now attempting to identify the precise region determining tissue specificity. In vitro, he has purified a general transcription factor called CBF which binds to CCAAT box motifs in both the a2(I) and a1(I) collagen promoters. This factor consists of two different peptides, CBF-A and CBF-B. Both factors have been purified and their cDNAs cloned, and both share homology in one segment to two yeast factors, Hap3 and Hap2, which also make up a multimeric transcription factor. Dr. deCrombrugghe also described the regulation of type I collagen by NF1, TGFb and TNFa. Information about collagen gene regulation will undoubtably be applicable to other eukaryotic transcription systems.
Dr. Nobuyuki Tanaka then summarized his work on regulation of the interferon-b gene. Interferons are cytokines that exhibit multiple biological activities on a number of different target cell types, and influence cell growth and differentiation. He described progress in the characterization of two transcription factors, IRF-1 and IRF-2, which are related and bind to the same cis elements in the interferon gene. Vectors expressing cDNAs encoding these factors were introduced into factor-negative embryonal carcinoma cells. These studies revealed that IRF-1 is an activator and IRF-2 a repressor of transcription. Dr Tanaka described new work on IRF protein modifications which regulate their activity, and suggested that ultimate control of interferon gene activity may be determined b regulation of these modifiers.
Dr. Shunsuke Ishii reviewed aspects of the c-myb proto-oncogene and its viral counterpart, which is truncated at the N and C termini. c-myb is unique in its ability to transform hemopoietic cells, and is involved in the proliferation and differentation of these cells. Dr. Ishii related that the myb gene product binds to a specific DNA sequence and helps regulate transcription of genes containing this sequence. He described the structural analysis of c-myb, showing that there are 3 functional domains: an N-terminal DNA binding region, an acidic transcriptional activation domain, and a C-terminal negative regulatory region. Dr. Ishii also showed that myb can transcriptionally control other important gene promoters such as c-myc and c-erbB2. He introduced a potentially novel regulatory region, the tryptophan cluster, that is essential for normal myb function. He finished by describing preliminary work on transgenic mice carrying the myb gene driven by a chicken b-actin promoter.
Trans-acting Transcription Factors
Dr. Jun-ichi Fujisawa continued the gene regulation theme by beginning this session with a discussion of HTLV-1 transcriptional regulation by the viral open reading frame factor p40tax. Tax has been shown to activate its own promoter, as well as cellular promoters of IL-2, GM-CSF, c-fos and others, suggesting that it plays an important role in abnormal proliferation of T cells. He mapped the tax responsive region to a 21 bp direct repeat enhancer element in the HTLV-1 LTR. However, tax does not directly bind to this enhancer element. To identify cellular DNA-binding factors that mediate tax trans-activation activity he cloned cDNAs that can bind to the 21 bp enhancer. These protein, called TREBS, contain leucine zipper structures and adjacent basic domains. Dr. Fujisawa described their potential role in signal transduction, and concluded with a discussion of the importance of cellular components in viral trans-activation.
Dr. Michael Karin presented a review of jun/fos and recent data on their interaction with glucocorticoid receptors. AP-1 is a widely distributed transcription complex composed mostly of products of the c-jun and c-fos proto-oncogenes. AP-1 stimulates transcription of many genes in response to ligand-or phorbol ester-mediated protein kinase C activation. Jun-jun and jun-fos dimerization is essential for transcriptional activation. Dr. Karin discussed the multiple levels of regulation of the AP-1 complex, including interactions between jun and fos, phosphorylation as a post-translational modifier, positive autoregulation of transcription by jun, transcriptional repression by both jun and fos and signal amplification by intercellular communication. He concluded with recent data on the negative interactions between c-jun and the glucocorticoi~ receptor. Cross talk between these two elaborate and widely used regulatory pathways establishes new and exciting prospects for future research.
Growth Factor and Targeting
Dr. Glenn Merlino described recent findings using transgenic mice to study the EGF receptor signal transduction pathway. Mice bearing a human transforming growth factor!
!!(TGF!!!) cDNA driven by the metallothionein promoter were generated. These mice developed tumors of the liver and breast, and exhibited abnormal development of the pancreas and mammary gland. He concluded that this factor can act in vivo as a mitogen and a morphogen, and can contribute to neoplastic transformation. Dr. Merlino also described a line of mice that overexpressed the EGF receptor exclusively in the testis. In this line the transgene integrated into and inactivated an important testis-specific gene, resulting in sterility in homozygous male mice. This was apparently caused by the disassembly of specific flagellar axonemal microtubules resulting in sperm paralysis. He concluded by discussing the use of this mouse line as a model for human sterility. Dr. Anita Roberts presented a large body of data on the regulation and function of transforming growth factor!
!!(TGF!!!). TGF!!!is a potent negative regulator of growth in most epithelial and hematopoietic cells. While TGF!!!can inhibit tumor growth, it can also indirectly stimulate the development of unresponsive tumors by stimulating formation of connective tissue and new blood vessels, while suppressing immune surveillance. Several levels of regulation were discussed. For example, both retenoic acid and tamoxifen induce TGF!!!secretion by a post-transcriptional control mechanism. She also described work on the promoter, which lacks a TATA box but contains AP-1 binding sites. The TGF!!! promoter is trans-activated by c-jun and c-jun expression is upregulated by TGF!!!. Dr Roberts also discussed possible regulatory relationships between TGF!!!and ras, and ptax40. Finally, she listed mechanisms by which cells can lose the ability of growth inhibited by TGF!!!: loss of TGF!!!receptors, inability to activate latent factor, and alterations in the TGF!!!signalling pathway. Discussion included future examination of TGF!!!in a number of different systems. Dr. Ira Pastan presented a progress report on the use of novel cytotoxic agents in the treatment of cancer and related diseases. He reviewed the approach, in which attenuated truncated forms of Pseudomonas exotoxin are linked to growth factors or other cell recognition proteins, or recombinant single chain antibodies. These chimeric proteins are made by expressing hybrid DNAs in bacteria. Dr. Pastan described recent data using chimeric proteins containing TGF!
!!, IL2, IL4, IL6, CD4 or antibodies against the IL2 receptor. He has achieved great success specifically in killing cells overexpressing the appropriate receptors or antigens on their surfaces. He concluded by conveying his impressions of cancer therapy in general, and describing how this new approach will be most efficacious. The future development of these toxin fusion reagents will undoubtably benefit cancer patients by significantly improving treatment. Closing Remarks
Drs. Ishii and Merlino closed the meeting by acknowledging the many excellent papers that had been presented, and conveyed their impression that the meeting had been extremely effective in disseminating scientific information and promoting the exchange of ideas. Participants in overlapping fields spent many fruitful hours during the meeting and at dinner discussing recent results, and those in more distant fields were able to learn about new areas and expand their scientific horizons. Exchange of information between Japan and the United States, two leaders in the field of cancer research, will continue to be of paramount importance to the development of the field. In addition, the participants agreed that a number of international collaborations were forged during this meeting, and unanimously urged that future cooperative efforts between researchers in the United States and Japan continue.
(3) Seminar on “Molecular Mechanisms in Carcinogenesis and Tumor Progression”
The US-Japan Cooperative Cancer Research Program meeting entitled "Molecular Mechanisms in Oncogenesis and Tumor Progression" was held from on January 17 and 18, 1991, at the Coco Palms Resort in Kauai, Hawaii. The meeting, which was organized by Drs. Takehiko Sasazuki and Stanley Korsmeyer, involved the participation of six Japanese and seven U.S. scientists.
The first session was entitled "Mechanisms of Negative Regulation." Dr. Arnold Levine of Princeton University initiated the session with a presentation of the functional role of the p53 gene as a tumor suppressor. He detailed the distribution of somatic mutations found in the p53 gene within tumor cells. There are some 103 examples of mutation of one allele and reduction to homozygosity by loss of the other allele. The somatic mutations focus within four predominant regions. These result in changes in the protein's ability to complex with HSP70 and also lead to marked prolongations of the usual 20 minute protein half-life. The inherited familial predisposition to cancer in the Li-Fraumeni Syndrome is found at positions 248/256, different from the more profound effector mutations found in sporadic somatic mutations. Importantly, Dr. Levine who was the first to demonstrate a tumor suppressor role for p53 now has generated an inducible system for p53 regulation that also produces morphologic reversion of transformed phenotypes. In other studies in which he generated a temperature sensitive mutant of p53 he noticed differential trafficking between the cytoplasm and the nucleus. At 32° the protein was located in the nucleus and the cells arrested at the G1/S boundary. Additional insights were provided into the interactions of human papilloma virus and p53. The E6 protein of HPV complexes with p53 and appears to activate a ubiquitin degradation pathway. Thus, for example, in cervical carcinoma it appears that p53 is in germline form but that the virus may be targeting this anti-oncogene product for degradation.
Dr. Ed Harlow of the Massachusetts General Hospital followed with a discussion of cell cycle control aspects of tumor suppressor genes. It was initially noted that anti-E1A immunoprecipitations identified the cyclin A as well as RB protein. This is part of a higher molecular weight complex together with a p130 and p107 protein. Furthermore, cell cycle specific phosphorylation of RB has been documented in which the Go stage is nonphosphorylated and is phosphorylated at G2. cdc2, p34 is capable of phosphorylating RB and has several classic cdc2 consensus sites for phosphorylation. Moreover, cdc2's association with other proteins including cyclin B varies throughout the cell cycle. The differential phosphorylation of RB changes its capacity to bind other proteins and may be intimately associated with the control of cell cycle progression.
Dr. Toru Akiyama of Osaka University presented work on the suppression of cell growth by c-Jun and c-fos. He has found evidence that the phosphorylation status of the c-fos protein changes its affinity for Jun. Jun fos heterodimers form readily in their nonphosphorylated state which can interact with TRE inducing gene transcription. In contrast, highly phosphorylated fos does not as readily interact and coprecipitate with Jun. The highly phosphorylated fos appears to be able to trans-suppress its own promoter leading to further decreased protein production. This intriguing biochemical system has the attractive biologic counterpart in the knowledge that TPA stimulation results in the underphosphorylation of at least one site on the protein.
Dr. Wen-Hwa Lee of the University of California in San Diego further detailed the molecular basis of tumor suppression by RB and p53 genes. Dr. Lee utilized four cancer cell lines that were lacking the RB gene which included retinoblastomas, osteosarcomas, prostate carcinoma as well as breast carcinoma cell lines. Wild type RB was transfected into those cells and they were characterized for changes in growth rate, colony formation, and tumorigenesis in nude mice. While there was some cell type variation in the parameters corrected the introduction of RB affected all three of these characteristics. Quite excitingly, Dr. Lee provided evidence for the identification of a p48 molecule which was a normal cellular protein that appeared to be a target site for RB interaction. This molecule represents a candidate for the long awaited cellular equivalent of the viral E1A protein.
Dr. Paul Polakis of Cetus Corporation in Emeryville, California, completed this section with the discussion of the GTPase activating proteins for Ras and K Rev. He has generated evidence that there are two functional forms of Gap. The original Gap which interacts and regulates p21 Ras, and a similar molecule that regulates the K Rev molecule which they entitle Rap. The Rap/Gap molecule is p88 in size and once again interacts with the GTP form of Rap. The cloned molecule has no homology to the classic Ras Gap nor NF-1 and does not regulate Ras. However, the original Ras Gap does appear to be able to interact with Rap in its GTP form. K Rev or Rap has been shown to suppress transformation by Ras and is approximately 20-fold more active than Ras. One intriguing possibility of this avenue of research is a regulatory interaction that might be manipulated. Perhaps Rap Gap functions to keep Rap in its GDP form which would free Gap for more effective regulation of p21 Ras. This interactive complex is further complicated by the discovery of the p190 protein that interacts with approximately half of the Gap proteins in transformed cells suggesting that it may be an inhibitor of the GTPase activity.
The Thursday evening session was entitled “Technical Approaches to Suppressor Gene Isolation”. Professor Mitsuo Oshimura of Tottori University discussed the role of micro cell mediated transfer and the identification of tumor suppressor genes. This technology assesses the capacity of isolated chromosomes in micro cells to suppress tumorigenesis when transferred to various carcinoma cell lines. Chromosomes implicated in various cancers include chromosome 18 in colon carcinoma, chromosome 1 in neuroblastomas, chromosomes 1 and 11 in fibrosarcomas, chromosome 11 in Wilms Tumor and chromosome 3 in renal cell carcinomas. Dr. Oshimura has concentrated on the chromosome I tumor suppressor gene by following the cytogenetics of revertants that lose their suppressor activity. Chromosome segment 1q12-13 is implicated by those studies.
Dr. Bernard Weissman from the University of North Carolina further extended the role of micro cell fusion to identify tumor suppressor genes in pediatric cancer. His work has identified linked tumor suppressor genes with p53 on chromosome 17 that are within several hundred kilobases. This appears to be the predominant locus operating in neuroblastomas on 17p. He has further defined a region on 11p that is responsible for the suppressor in Wilms tumor by using irradiation reduced micro cell hybrids. This would map to 11p14-11p15 region different from the previous isolated zinc finger candidate at 11p13. He finds a second tumor suppressor locus on chromosome 11 between 11q23 and the telomere that is capable of suppressing peripheral neuroepitheliomas and fibrosarcomas. This is an intriguing location and raises the outside possibility that it might be related to the Ewing sarcoma translocation site.
Dr. Stan Korsmeyer from Washington University in St. Louis concluded this session with a discussion of the use of YAC cloning to identify chromosomal translocations and deletions that were a considerable distance from known genes. This group had developed uniformly applicable technologies to confirm the authenticity of YAC isolates, to localize genes internally, to restriction map YACs and finally to rescue the distal ends of the genomic inserts. The rescued ends of the genomic clones were isolated by inverted polymerase chain reaction technique and then converted to sequence tagged sites. This allowed the creation of PCR primers that enabled the rescreening of the YAC library and marching to be accomplished. By utilizing these methodologies some 2500 kilobases of chromosome segment 18q21 had been physically cloned and mapped. This converted a probe-poor region into a probe-rich area now well equipped to search for human disease loci. Meiotic recombination had also been used to assemble a single YAC containing the entire Bcl-2 locus which spans some 230 kilobases. Drug selectable markers had been inserted into the YAC and it successfully transfected into target cells to begin to study the regulation and deregulation of a large gene in its entire genomic context.
The final session on Friday morning was entitled "Identifying Tumor Suppressor Genes in Human Cancer." Dr. Takaaki Sato of the Japanese Foundation for Cancer Research in Tokyo initiated the program with a talk concerning the multiple genetic alterations found during progression of breast cancer. He had used loss of heterozygosity Loh studies in some 600 breast cancer specimens. The candidate loci that most frequently turned up were chromosomes: 3p, 11p, 16q and 17p. Correlations that surfaced included a relationship between 17p Loh and a Loh at 13q in some of the most malignant histologies. Provocatively further mapping with multiple RFLP markers located on 17p indicates that the region with the highest incidence of Loh appears to be distal to the p53 gene. This emphasizes the importance of multiple probes for Loh analysis in order to find the responsible loci.
Dr. Jun Yokota from the National Cancer Center in Tokyo presented further data on the loss of heterozygosity concentrating on lung carcinoma. Both small cell lung carcinoma as well as adenocarcinomas implicated the involvement of tumor suppressor genes at : 3p, 13q and 17p. Individual cases showed tumor progression in which 13q losses followed 3p losses. He also noted the presence of more Loh for 13q and 17p in brain metastases versus the initial primaries. To further dissect the 17p abnormalities he used PCR generated fragments in single stranded conformation polymorphism (SSCP) analysis. Up to 60% of metastases and nearly 90% of established cell lines showed mutations in p53 and included a wide variety of abnormalities.
Dr. Andrew Feinberg of the University of Michigan presented data on the identification of multiple Wilms tumor suppressor genes. Wit-1 and Wit-2, one of which is identical to the previously described zinc finger protein are at 11p13 while another provocatively localizes to 11p15. He noted that the WAGR syndrome is associated with intra-lobar nephrogenic rest whereas the Beckwith-Wiedemann syndrome is associated with peri-lobar nephrogenic rest. The WAGR tumors occur at young ages while the Beckwith-Wiedemann syndromes are older. The ILNR and WAGR type of tumors may well have loci that map to 11p13; whereas, loss of heterozygosity studies both in Beckwith-Wiedemann syndrome and sporadic Wilms tumor argue that these PLNR type tumors map to 11p15.
Dr. Takehiko Sasazuki from Kyushu University followed with a discussion of the detailed analysis of chromosome 5q abnormality in familiar polyposis coli. This is an autosomal dominant disorder with approximately a 95% penetrance. Importantly Dr. Sasazuki has shown that the micro cell transfer of human chromosome 5 into the SW620 target which has Loh for chromosome 5 resulted in a decreased growth rate and extended latency of tumor generation. Curiously Dr. Sasazuki has shown that the other associated abnormalities in tumors from FPC and non-polyposis coli (NPCC) are similar. Both show Loh on: chromosome 14, 18, and 22. Moreover p53 abnormalities were also noted in FPC as well as NPCC.
Dr. Masao Sasaki completed the session with a discussion of the origin of loss of function mutations in tumor suppressor genes. He documented the retention of the paternal origin allele for chromosome 13 when it was reduced to homozygosity. He presented data that indicated that the retinoblastoma mutations were not related to a paternal age effect. This argues against a pre-meiotic male abnormality as the cause for cancer genomic imprinting. His data argues for an unequal involvement of alleles in which the first somatic event following the deletion preferentially involved the paternal origin chromosome. His data are very striking and argues strongly for a role of parental imprinting in the selection of mutated alleles in a number of oncogenes.
This meeting was of uniformly high quality throughout with the presentation of large bodies of previously unpublished data. This stimulated very vigorous and widespread discussions which can be expected to prompt further investigations.
SEMINAR AGENDA AND PARTICIPANTS
(1) Seminar on “Antigen presentation and immune cell interaction”
Osaka, Japan, November 8-10, 1990
AGENDA