REPORTS ON SEMINARS

(1) Joint Seminars on “Cellular and Molecular Mechanisms of Oncogenesis and Cancer Immunity” and “Regulatory Elements in Normal and Transformed Cells”
The U.S.-Japan Cancer Research Cooperative Program Meeting was held in Seattle, Washington, at the Westin Hotel, November 10-12, 1986. This meeting represented a combination of two groups, one headed by Drs. Richard Hodes and Toshiyuki Hamaoka concerned with tumor immunology. The other group was headed by Drs. Tadamitsu Kishimoto and Stanley Korsmeyer, and focused on molecular immunology and molecular diagnosis.
This meeting involved participation of 15 U.S. and 15 Japanese scientists and was divided into six major topic areas.
The first session began on Monday morning, November 10, with the consideration of immunoglobulin genes and immunoglobulin receptors and their gene regulation. Dr. Philip W. Tucker of the University of Texas Southwestern in Dallas, presented a review of his work on the regulation of murine immunoglobulin heavy chain gene transcription. He focused upon the identification of upstream sequences 5’ to the variable regions and demonstrated consensus sequences responsible for the binding of regulatory proteins. He demonstrated an important asymmetry in terms of the coding versus non-coding strand within the upstream regulatory sequences known as the octamer. The data indicated that there were probably at least two species of proteins involved in the binding to the downstream octamer within the immunoglobulin enhancer region. Dr. Tucker then turned his attention to the regulation of the secreted versus membrane forms of immunoglobulin transcripts. In the setting of the mu membrane versus secreted decision, he presented data favoring a regulatory choice at the level of transcriptional termination in RNA cleavage rather than at a later polyA addition or truly alternative RNA splicing step.
Dr. Takeshi Watanabe of Kyushu University followed with further dissection of regulatory proteins involved in human immunoglobulin gene expression. Dr. Watanabe successfully exploited his very unique RBC ghost transfer system to fuse B cell nuclear factor proteins into fibroblast L cells. He showed that he could successfully transfer the transactivating factors into L cells and obtain effective immunoglobulin gene transcription. This result was entirely dependent upon the immunoglobulin enhancer region within the construct, as enhancer negative constructs displayed no positive effect from the B cell factors. He then successfully fractionated using an S-300 column the functional binding activities. Gel retardation assays were able to demonstrate that fraction 2 bound to the enhancer core sequence shared with SV40, while fractions 3 and 5 had distinct binding activity for the proximal and the distal octamer, respectively. This approach is very promising and provides a functional readout on specific proteins that can be identified in classic gel retardation assays.
Co-Organizer Dr. Tadamitsu Kishimoto of Osaka University completed the morning session with a discussion of his cloning and characterization of the CD23 antigen which proves to be the Fc-epsilon receptor on B cells. His group created several monoclonal antibodies capable of inhibiting the binding of IgE to a receptor positive cell. They were able to immunoprecipitate a 45 Kd protein corresponding to that receptor and then successfully transfected high molecular weight DNA from the cells into L cells and got receptor expression. They subsequently cloned the Fc-epsilon receptor by capitalizing on an L cell subtraction and oligonucleotide probe from the protein sequence. The cDNA encoded a 321 amino acid protein that was shown by expression studies to recreate the Fc-epsilon receptor function. This function has an interesting structure like some other receptors with an extremely cysteine-rich extracellular domain in which the C-terminus is outside and N-terminus is inside the cell. Important differences were also noted in the RNA species within monocytes versus B cells, and this receptor is clearly different from the epsilon receptor present on mast cells.
The following session was “Gene Expression in Lymphoid Cell Activation.” Dr. Kazuo Sugamura from Tohoku University presented a very provocative IL-2 dependent T cell line variant that increased its growth rate following phorbol ester stimulation with TPA. This result appeared to be independent of a protein kinase C pathway in that the general level of phosphorylated proteins and typical PKC target phosphorylation sites did not appear to change. There is a possibility of a PKC independent activation pathway which might possibly relate to the more than 3 cross hybridizing genes.
Dr. Roger Perlmutter of the University of Washington in Seattle then followed with a discussion of several tyrosine kinases important in lymphocyte differentiation that his laboratory has isolated and characterized. Initially, the laboratory concentrated on the first isolate now referred to as LCK which is located on the short arm of chromosome 1 at lp32-35 potentially next to the c-fgr gene to which it bears some homology. This gene is turned on predominately early in the activation of T-lymphocytes and can normally be seen in the cortex of the thymus. The laboratory has also isolated and analyzed a gene expressed in B cells as well as some monocytes which has a very similar protein kinase and is referred to as BCK.
Dr. Kathleen Kelly of the National Institutes of Health completed the morning session with a discussion of the expression of the nuclear located oncogene family of foc, myc, and myb during lymphocyte activation. She noticed that PHA induced C-fos but that interleukin-2 did not. In contrast, both IL-2 and PHA induced C-myc. Cycloheximide was noted to give a super induction of myc and fos, and another protein inhibitor was shown to super-induce C-myb. After this initial kinetic work, she turned to the isolation of new genes that would be present following T cell activation and made double subtraction libraries of T cells activated with a combination of PHA, PMA and cycloheximide to assure superinduction of certain genes. This library was screened with a probe that was a subtraction product of an activated T cell minus quiescent T cell RNA. She appears to have at least 25 clones that are unique genes. This set of clones holds a promise of containing genes which may not have been previously recognized by other approaches.
The third session dealt with T cell receptors and T cell repertoire expression. Dr. Michael Brenner of Harvard University described recent studies clarifying the nature of a T cell receptor distinct from the alpha/beta “conventional” T cell receptor. The second receptor, the gamma/delta dimer, was found to be expressed on cells which do not express the alpha/beta molecules. At least two forms of the gamma/delta receptor appear to exist, one consisting of non-disulfide linked 55KD and 40KD chains, and a second consisting of disulfide linked 38KD and 35KD forms. Studies of the functional role for these receptors are in progress. Dr. Charles Janeway of Yale University has studied the alpha/beta T cell receptor through the use of a large panel of monoclonal antibodies directed at different epitopes on the T cell receptor molecule. Antibodies differed in their ability to activate T Cells, and in their association with the CD3 molecule. These studies also suggested a differential relationship between the CD4 molecule and different determinants on the T cell receptor complex. Dr. Richard Hodes of the National Institutes of Health described studies of the minor lymphocyte stimulating (Mls) system. The products of this system are involved in the activation of a high proportion of T cells. Conventionally, the Mls system has been regarded as a single gene, multi-allelic polymorphic gene system. The genetic and clonal analysis presented at this meeting indicated that, in contrast to these presumptions, Mls appears to consist of at least unlinked genes, with no definitive evidence for polymorphism at either gene locus. The implications of these findings for Mls function in T cell recognition were discussed. Dr. Alfred Singer of the National Institutes of Health described studies in which the repertoire for recognition of mutant class I MHC determinants was analyzed. Findings indicated that mutant class I determinants were recognized in the context of self I-A determinants. A model was presented suggesting that T cell repertoire selection is based upon intrathymic recognition of both self restricting element and a nominal self antigen. Dr. Katsuo Kumagai of Tohoku University described the T cell response in autologous mixed lymphocyte reaction. The nature of the lymphokine response to self antigens was quite distinct from that involved in mixed lymphocyte responses to alloantigens. Syngeneic or autologous responses led to induction of IL-3 but without detectable IL-2 or interferon gamma. Dr. Jay Berzofsky of the National Institutes of Health then described the features of antigenic determinants which are associated with immunodominance. Features included the nature of Ia specificity for a given antigenic site as well as factors intrinsic to the antigen, most prominently the characteristic of amphipathicity, which correlated highly with immunodominance in a number of antigenic systems.
The Tuesday morning session on November 11 began with a discussion of lymphokines and lymphokine receptors. The first speaker was Dr. Tadatsugi Taniguchi of Osaka University. He discussed the genetic regulation of both IL-2 and the IL-2 receptor. He showed that there were 3 to 5 upstream start sites or promoters for the IL-2 receptor. In co-transfection assays using interleukin-2 or the interleukin-2 receptor upstream sequences he found complimentation by the introduction of the pX or Tat region of HTLV-I. He determined that only the P40 protein is important in terms of transactivation. These successfully co-transfected Jurkat T cells interestingly would show a synergistic increase in activation with the addition of anti-T3. This experimental model only worked within T cells and not within B cell target cells.
Dr. Junji Yodoi of Kyoto University followed with a discussion of the factor he has termed ADF isolated from leukemic cells. This factor is capable of inducing interleukin-2 receptors, especially those of high affinity on T cells. He showed that separate pathways for forskolin (an A kinase) also were capable of inducing interleukin-2 receptors of high affinity. He then turned his attention to the cloning and characterization of the Fc-epsilon receptor. He ended with a discussion of CD28 called Kolt-2 antigen, a very large molecular weight antigen found at low levels in resting T cells but increasing dramatically with activation and appearing before interleukin-2 receptor.
Dr. Toshimitsu Uede of Sapporo Medical College completed the discussion of the interleukin-2 receptors with a characterization of an antibody reactive with rat T cells known as 5C6-F4 which appears prior to the interleukin-2 receptor. This antibody inhibits proliferation and recognizes a high molecular weight greater than 100 Kd molecule. He then turned to the exploitation of a human system in which he had an adult T cell leukemia line that seemed to express only low affinity receptors. He then took interleukin-2 and crosslinked with DDSSP and by 2D-gel electrophoresis felt that there was demonstration of an associated 75 Kd protein.
The second half of the morning session was initiated by Dr. Toshiaki Ohsawa of Tokyo University. He presented his studies concerning human macrophage activating factors. He discussed two factors, maf-c present at one day and maf-c6 present at the sixth day. He was able to purify these factors by column chromatography and found fractions that were synergistic in their action. The first day factor was species specific, and the sixth day factor was not specific. He constructed liposomes in which he placed the macrophage activating factor internally together with gamma-interferon and on the surface put an A375 melanoma antigen. This proved quite effective in terms of preventing subsequent tumors when melanoma was injected into recipients. He also had some impressive data showing the effectiveness of this approach in treating mice already bearing melanoma.
The subsequent paper was presented by Dr. Kiyoshi Takatsu of Kumamoto University. He presented his exciting data concerning the molecular cloning of T cell replacing factor. This factor has also been known in the past as B cell growth factor-2 and has the currently proposed name of interleukin-5. TRF is a 45-60 Kd size glycoprotein with a reduced subunit of 18 Kd. This factor was cloned by using an SP6 RNA expression library in which 5x10⁴ cDNA clones were linearized and subjected to SP6 polymerase and oocytes injected. He found one clone that had the TRF activity. Its sequence shows no extensive homology with the other lymphokines and in vitro translation revealed a backbone of 12 Kd. An antibody was made to the product, and it clearly inhibited all TRF activity in other assays.
The final speaker in the morning was Dr. Toshio Hirano of Osaka University who presented his studies on the cloning of the human BSF2 gene. BSF2, also known as B cell differentiation factor, induces secretion of immunoglobulin in Epstein-Barr virus lines and activated B cells but induces no proliferation. N-terminal sequence was determined and internal fragments, 7 in number, were also sequenced. A cDNA Iibrary was made in the SV40 plasmid vector and screened with oligonucleotides. From the determined protein sequence the positive clones were placed in cos-7 cells, and the predicted product made. There is a 212 amino acid precursor that is cleaved to a 184 amino acid product. It has a 3’ untranslated region once again rich in destabilizing AT stretches and bears a 25% homology with G-CSF. The recombinant BSF2 product induces IgG production in tonsilar B cells following activation with staphylococci but there is no proliferation.
This session continued on Tuesday afternoon, when Dr. Robert Coffman of DNAX Corporation discussed the role of lymphokines in mediating T helper cell function. The multiple biologic activities of IL-4, including both T cell and B cell activating functions were described. In addition, the division of T helper cells into two subsets on the basis of patterns of their lymphokine production was described. Dr. Ellen Vitetta of the University of Texas, Southwestern Medical School, Texas, further detailed the functions of IL-4 as both a T cell growth factor and a B cell activation and switch factor. Available data were then synthesized into an hypothesis regarding the role of lymphokines in T-T and T-B cell interactions.
The final session on Tuesday, November 11, concerned tumor antigens and oncogenes. The first speaker, Dr. Mark I. Greene from the University of Pennsylvania, discussed his studies of the neu oncogene. This 185 Kd glycoprotein has a classic tyrosine specific kinase domain. It has been shown that nucleotide number 2012 is mutated in the oncogenic form resulting in a change from a valine to a glutamic acid in the transmembrane region. He showed a very interesting pathway of internalization of p185 antibody that could be induced with antibody and curiously decreased the new synthesis of pl85. In addition, some of the antibody would actually block colony formation when the neu oncogene was transfected into cells. Some of these antibodies that have been generated also give partial protection to challenge with tumor cells. This antibody recognizes only the neurons within brain tissue and has its onset of expression about day 15 of development exactly at the time when carcinogens are effective in mutating the neu oncogene. Studies of human tumors have found that approximately 7% of adenocarcinomas and up to 40% of breast carcinomas may amplify this cellular oncogene.
The following speaker was Dr. Noriyuki Sato of Sapporo Medical College. He discussed induction and identification of a new cell surface associated antigen following transfection of the ras oncogene. He developed a monoclonal antibody against a EJ ras transformant that proved to recognize something other than the classic p21 ras product. He called this antigen 109 and it is an 86 Kd antigen which has affinity for lectins. It is occasionally found on other tumor cells, particularly in some sarcomas, although their ras configuration is not certain. He has not seen it normally expressed in adult tissue, and it is not induced following transfection of c-myc. Characterization of this protein and its gene could provide a very interesting example where the point mutation of a single amino acid in ras resulted in the induction of this species.
Dr. Masaru Taniguchi of Chiba University followed with the genomic cloning of a DNA segment controlling the expression of a mouse melanoma antigen. The melanoma antigen he has identified is referred to as GM3, and the approach to clone this was a genomic transfection system. Partial MboI digested DNA in the size range of 35-45 Kd was ligated into a vector and then by protoplast fusion was placed into target cells. These subsequent cells were screened with the antibody to the GM3 antigen by continually taking the most positive cells as determined by flow cytometry and recloning and regrowing those. The genomic transfected DNA was recovered by using a phage rescue in vitro packaging approach made possible by that vector. Three isolates were found, one of which, PD2-7, did cause the induction of antigen when transfected and appears to have a primary transforming capacity in L cells as well as NIH 3T3 cells. These subsequent cells were then shown to be tumorigenic within nude mice. He did subsequent northern analysis and identified several RNA species, and has several independent cDNA isolates of this gene.
The last speaker of the day was Dr. Stanley Korsmeyer from Washington University, who discussed the topic of the “Lineage Association of Chromosomal Translocation.” He concentrated on the t(14;18) translocation typical of mature B cell follicular lymphomas. He showed that the mechanism of recombination was the insertion of a staggered double-stranded break within a B cell associated gene (Bcl-2) from chromosome 18 into the diversity and the joining region segments on chromosome 14. Thus, this translocation occurs early in pre-B cell development at the first step of immunoglobulin gene joining the D to J junctura. The translocation event interrupts the 3’ exon of the Bcl-2 gene resulting in a fusion transcript with the immunoglobulin heavy chain locus. This leads to a marked deregulation of this gene and inappropriately high levels of this transcript within translocated versus non-translocated cells. He also showed that this same translocation can occur in T cells that have initiated D/J recombination, although the most common site of translocation in T cell neoplasms are the T cell receptor genes. He is exploring a family that has an inherited translocation at the alpha T cell receptor locus 14q11 that subsequently develops T cell neoplasm in a prospective fashion.
The sixth and final session was held on Wednesday morning and dealt with clinical and preclinical tumor immunology. Dr. Yoshiyuki Hashimoto described the properties of lymphokine-activated killer (LAK) cells. LAK cells were derived from spleen, tumor infiltrates, thymocytes, or nude mice. These LAK cells were then characterized with respect to phenotype and spectrum of cytotoxic activity. The relationship of these cells to the T cell lineage was demonstrated. Nagahiro Minato described the reproducible establishing of large granular lymphocyte (LGL) lines. The potential function of IL-3 in their growth and differentiation was suggested. Rearrangement of the T cell receptor alpha and beta as well as gamma genes was identified in multiple LGL clones, which also expressed the Thyl⁺ lines.
The requirements for T cell recognition in vitro and in vivo antitumor responses was discussed by Dr. Philip Greenberg. In adoptive transfer in vivo experiments donor T cells were shown to mediate irradiation of tumor in the murine model. T cells worked optimally in cooperation with cytoxan or other antitumor agents. Roles for both L3T4⁺ and Lyt2⁺ cells were established. A proposal for human application of this model was described. Dr. Toshiyuki Hamaoka described tolerance induction and recovery of responsiveness to tumor specific antigens. In vitro and in vivo responses were studied in both bone marrow chimeras and after tolerization of intact animals. Acceleration of recovery of the antitumor response was achieved using a system of hapten immunization and hapten-tumor cell conjugates. Within this model, enhancer antitumor in vivo responses were generated, with the apparent participation of hapten-specific T helper cells. Dr. Masuo Hosokawa described means for using local (intra-tumor) IL-2. The use of pluronic gels to give sustained local release of IL-2 was effective in increasing anti-tumor immunotherapy effects. Dan Longo described the current status of studies in the use of monoclonal antibodies for cancer diagnosis and treatment. The technical difficulties and current limitations in immunodiagnostic procedures were emphasized. Several new experimental approaches to tumor immunotherapy were described. These include the use of antireceptor antibodies for down-regulating the growth of T cell tumors in the mouse, where dramatic cures have been seen.
This joint meeting of immunology and diagnosis was judged by all participants to be an especially productive format. Discussions were extensive and highly useful. In particular, the opportunity for interaction between immunologists and molecular biologists concerned with similar areas of cell biology and tumor biology were judged to be extremely useful. It is anticipated that periodic joint meetings between these two program areas will be held.

(2) Seminar on “Oncogenes in Relation to Developmental and Transcriptional Control”
A U.S.-Japan Seminar, as a part of the U.S.-Japan Cooperative Cancer Research Program, was held at Sheraton Makaha Hotel at Waianae, in Oahu Island, on February 12-15, 1987. A U.S.-Japan meeting on “Oxygen Radicals in Cancer” last year was also held there (Jpn. J. Cancer Res. (Gann) 77, 843-848, 1986). U.S. participants were: Dr. James E. Darnell (The Rockefeller University, U.S. organizer), “Gene Expression Specific for Liver Differentiation,” Dr. David Baltimore (Whitehead Institute for Biomedical Research, MIT), “Regulation of Immunoglobulin Genes,” Dr. J. Michael Bishop (University of California, San Francisco), “Oncogenes and Cellular Differentiation,” Dr. Robert Tjian (University of California, Berkeley), “Transcriptional Regulation Factors and Enhancers,” Dr. Robert G. Roeder (The Rockefeller University), “Purification and Function of Transcription Factors” and Dr. Patrick H. O’Farrell (University of California, San Francisco), “Role of Engrailed Locus in Gene Control in Drosophila.”
Japanese participants were: Dr. Masami Muramatsu (The University of Tokyo, Japanese organizer), “The Molecular Basis of Species Specific Transcription of Mammalian Ribosomal RNA Gene,” Dr. Tasuku Honjo (Kyoto University), “IL-2 Receptor: Structure, Function, Expression and Its Abnormality, “Dr. Yoshiaki Suzuki (National Institute for Basic Biology),” Tissue-Specific Transcription Enhancement of the Fibroin Gene Characterized by Cell-Free Systems, “Dr. Hideya Endo (Medical Institute for Bioregulation), “Sequences Abundantly Expressed in Tumor and Growing Cells,” Dr. Takeshi Watanabe (Medical Institute for Bioregulation), “Regulation of Immunoglobulin Heavy Chain Gene Expression,” Dr. Yoshiaki Fujii-Kuriyama (Cancer Institute), “Structure and Regulation of Cytochrome P-450 Genes” and Dr. Kenshi Hayashi (National Cancer Center Research Institute), “Mechanism of Regulation of c-myc Gene.”
The objective of this particular meeting was to put together the present knowledge of the basic mechanisms of eukaryotic gene transcription and investigate the relevance of the transcriptional control to malignant growth of the cell. The topics represented by the above titles included a variety of approaches to better understanding of the regulatory mechanisms involved in eukaryotic gene expression. In particular, a number of enhancers and enhancer-like elements and their specific transacting factors were discussed in detail.
Dr. Muramatsu reviewed the present status of knowledge concerning the molecular mechanisms of transcription initiation by RNA polymerase I on ribosomal RNA gene. There are at least two (probably more) factors beside RNA polymerase I that are necessary for formation of transcription initiation complex on ribosomal RNA promoter. A factor designated TFID binds first to the promoter region, followed by a second factor TFIA and RNA polymerase I. The former two factors remain bound to the promoter while the polymerase repeats transcription from the complex many times. Since TFID has a species specificity, human-mouse chimeric genes constructed by gene engineering have been used to determine the recognition site by TFID on the promoter. A chimera having a mouse sequence only between -14 and -32 in the human promoter could be transcribed well (~90% of wild type mouse promoter) by mouse extract. In accordance with these results, -12 to -40 region was found to be protected well by partially purified TFID against DNase I. Certain upstream sequences were also protected by the footprinting analysis. Whether these multiple footprints are all due to TFID is not known at present,
Dr. Tjian described the transcription regulatory factors and their interactions with various promoter and enhancer elements using different systems. A Drosophila homeotic gene, Ubx, is activated dramatically at 0.5h to 8-12h after fertilization. When he challenged extracts from embryos of different stages, multiple footprints were obtained in the upstream and downstream of the transcription start site. Some footprints were ubiquitous but others were unique for certain stages. Then, he summarized the interaction of purified SP1, a transcription enhancing factor, with the promoter region (mainly 21 bp repeats) of SV40. He purified SP1 by sequence specific DNA chromatography followed by SDS gel electrophoresis. These proteins could be renatured and used for footprinting. This type of purification was applied with another transcription factor, AP1, whose binding site is between 21 and 72 bp repeats. This same sequence is also found in the basal level enhancer (BLE) elements that are present twice in the upstream region of metallothionein IIA gene. Another factor AP2 binds to multiple locations in SV40 control region. For instance, AP2 binds with sequences in 21bp repeat but on separate sites from SP1. There is neither competition nor synergism between SP1 and AP2. T antigen inhibits AP2 binding to 21 bp repeat but not the binding of SP1 to that region. T antigen also inhibits AP2 binding to multiple sites in metallothionein gene promoter and inhibits transcription, thereby showing an anti-enhancer activity. Then, why so many factors? Dr. Tjian speculates that different factors may act in different cells or conditions to increase transcription and multiple factors may reflect the efforts of the virus for host range expansion. In fact, only SP1 is required when transcription is tested in vitro. He recently obtained a partial cDNA clone of SP1 and detected mRNA of 8.2 Kb long by Northern blot analysis. Interestingly, a part of the predicted sequence had considerable homology to TFIIIA, having 3 zinc finger structures.
Dr. Roeder reviewed the transcription factors of RNA polymerase II and III, dividing those into basic (common) factors and gene (group) specific factors. RNA polymerase III system is now relatively clear. TFIIIA is required only for 5S RNA. TFIIIB and C are required for all known polymerase III systems. Their promoters are located inside the gene region (transcribed region), and the interactions with those factors are beginning to be understood. During adenovirus infection, the VA1 RNA gene is activated by the increase of TFIIIC. For the analysis of transcription by polymerase III, adenovirus major late gene, Drosophila heat shock protein (hsp) gene and histone H4 and H2B genes are used. General factors, IIA, IIB, IID and IIE, are identified as needed for accurate and efficient initiation of transcription. TFIID is apparently a TATA-box binding factor, but the fingerprint is different from one gene to another. For hsp 70, only TATA-box was protected, whereas other downstream sequences were protected for other genes. For H4 gene, a downstream sequence is required for TFIID to work, though this can bind with TATA-box in the absence of the downstream sequence. He is also working on B-cell specific promoter element and transacting factors. He found, in a histone H2B conserved element, a 15 bp repeat containing an octanucleotide common to immunoglobulin octamer element. Mutants of H2B octamer eliminate B-cell specific transcription of this gene. Protein footprints were found on normal octamer with a B-cell extract. There are two binding factors, OBF-B2 and OBF-B3. B2 is 90 Kd and ubiquitous, including HeLa cells and stimulates transcription of H2B gene, whereas B3 with a Mr 60 Kd is present in B-cells and enhances the transcription of IgK gene. Although B2 and B3 bind with IgK and H2B gene regions, respectively, they do not stimulate transcription of those genes. The nomenclature of these factors is not established, and participants agreed that Dr. Roeder’s factors B2 and B3 corresponded to Dr. Baltimore’s A1 and A2.
Dr Suzuki reviewed the present status of the investigation on tissue specific transcription regulation by using fibroin gene system. There appear to be three hierarchies in transcription control: basic, species- and tissue-specific. For instance, a fibroin gene could be transcribed in a HeLa extract at a certain level but in this case only TATA-box is required; i.e., 5’-delta-31 5’-deletion mutant having upstream sequence up to -31, has an activity. To support a better transcription in extracts from the silkworm, Bombyx mori, (e.g., ovarian tissue middle silk gland, and an embryonic cell line) 5’-delta-72 is necessary; this enhancement is supported by species specific and constitutive type factor(s). And a further upstream sequence 5’-delta-238 is required to get the tissue specific enhancement in posterior silk gland extract. He concludes, therefore, that a region, -73 to -238, is a tissue-specific “enhancer-like” element This enhancer is not typical for this reason: although this region can be functional even when inverted, it cannot be moved too far upstream or downstream of the transcription start site. The posterior silk gland extract has been fractionated by a phosphocellulose column into A-D fractions and transcription machinery reconstituted. Fraction D could stimulate the transcription of Adenovirus major late, sericin, chorion genes besides the fibroin gene. Therefore, it seemed to contain a basic factor. Addition of fraction D to a HeLa cell extract exhibited a tissue-specific enhancement with the 5’delta-238 mutant. He suspects that fraction D contains also tissue-specific enhancing factor.
Dr. O’Farrell, after reviewing briefly the Drosophila genes that are related to morphogenesis, discussed the possible function of the en (engrailed) gene based on his recent data. A combinatorial control appears among this family of the gene. He showed a model in which a limited number of regulatory elements could control a complex pattern of expression of a large number of the gene by combinatorial regulation. A helix-turn-helix structure is a characteristic feature of these proteins but helix 3 is most conserved among different organisms from human to sea urchin. He is investigating the binding characteristics of these both with gel shift and DNase I footprinting, using different fusion proteins. Engrailed protein appears to make a homodimer by antiparallel alignment, but whether a heterodimer with other proteins may be formed is not known.
Dr. Watanabe used L cells transfected with rearranged human IgG gene for detection of transactivating factor(s) for this gene. When nuclear extracts from myeloma cell lines were introduced into those cells by red blood cell ghost fusion procedure, they could induce human IgG gene transcription. This reaction was enhancer dependent. The inducing factor has been partially purified on HPLC(TSK-3000) and heparin-Sepharose column. Although both fractions 3 (Mr ~ 60-100 Kd) and 7 (Mr ~ 20 Kd) contained inducing activity, a positive gel-shift with enhancer element was found only with fraction 3. He identified the DNA sequences to which protein(s) with inducing activity appeared to bind and was trying to purify them with a DNA affinity column. He also purified to a certain extent the protein(s) bound to octanucleotide in the promoter region. But, proteins with IgG gene-inducing activity did not contain the octamer-binding protein. Further purification of this inducing factor(s) is now in progress.
Dr. Baltimore reviewed the recent progress in immunoglobulin gene expression, including his newest data. Light chain enhancer is much simpler than heavy chain control region. A octamer sequence present at 70 bp upstream of the transcription start site is very important and works in a highly tissue (B-cell) specific manner. There are at least two nuclear factors, NF-A1 and NF-A2, that can bind with this sequence, causing two bands of gel retardation NF-A2 appears to be lymphoid specific. The sequence of octamer appears very rigorous because one base change alone could eliminate the competition capacity in this assay. There are three regulatory segments in IgH, and the rearranged gene could express itself 10 times in B cells over 3T3 cells, even if the enhancer is removed. Different nuclear factors (NF’s) interact with various enhancer motifs in mu enhancer (mu E1-4, etc.). He also provided some new data on a nuclear factor which binds to a site in the enhancer (NF-kB). NF-kB appears to be required for activation of k-gene transcription, and extracellular ligands (such as LPS) can activate the factor through post-transcriptional modification. For example, TPA activated NF-kB in B cells, T cells and even HeLa cells, suggesting the phosphorylation by protein kinase-C may be involved in activation of NF-kB. He also showed that HTLV-III contains the sequences homologous to NF-kB binding site.
Dr. Honjo described the IL-2:IL-2 receptor (IL-2R) system as a basis of antigen-specific clonal expansion of lymphocytes. As to transcription regulation, a region between -100 and -300 is identified as required for lymphokine-dependent induction in YTC₃ cells. In vitro transcription system was established, and MT-1 cell extract was found to contain some transacting factor(s) for tissue-specific expression of IL-2 receptor promoter although it did not contain pX products. Importantly, ATL cell lines established in culture are different from major clones in patient’s blood, necessitating a new viewpoint in the pathogenesis of ATL. To analyze low and high affinity IL-2R’s, he used mouse T cell lines to which human IL-2R gene and its mutants were transfected, and function of mutant IL-2R was examined after blocking all the endogenous IL-2R by anti-mouse IL-2R antibody. These studies suggest the presence of the second chain which is designated a “converter” that is involved in converting the low affinity IL-2R to a high affinity one.
Dr. Bishop reviewed a family of genes coding for protein tyrosine kinase in various organisms, including Saccharomyces, Drosophila and mammals. Src expression increases several fold during differentiation of HL60 cells into either granulocytes or monocytes. These tyrosine kinase genes, including src, fms, lck and fes/fos, are preferentially activated in hematopoietic cells and also in brain cells. Although a number of protein are phosphorylated by these enzymes, nobody knows what phosphorylation is crucial for differentiation and possibly for malignant transformation. There are two protein kinase genes in S. cerevisiae but only one in S. pombe. In Drosophila, raf, myb, erb, src, fps and abl are identified. Src is expressed highly from the early stages of development, particularly in early brain. He also referred to the regulation of myc gene where a negative control element, or silencer, is found between -100 and -353 having homology with a regulatory region of beta interferon gene. In the positive control region, there are two NF1 binding sites, both locating near DNase hypersensitive sites.
Dr. Endo isolated 30 cDNA clones whose sequences were abundantly expressed in hepatoma cell lines but not in normal liver cells by using differential hybridization. They comprise both unique and repetitive sequences. Corresponding to the latter, two genomic clones, pRAL6 and pRAL10, were identified. Both sequences are expressed not only in many tumor cells but also in embryonic primary culture cells. They have a common region homologous to retrovirus LTR. However, the sequence of downstream half of its U3 region is different. pRAL6 is derived from a complete type endogenous virus, and pRAL10 is from a solitary type LTR. S-1 mapping shows that it is transcribed from the cap site and nuclear run-on assay indicates that the transcripts exist in AH60C but not in rat liver. Southern blot analysis shows that RAL6 provirus may be about 7 Kd long. 300 copies of proviral and 3000 copies of solitary sequences exist in rat genome.
Dr. Hayashi presented data on the regulation of c-myc during liver regeneration. In the rat, cmyc mRNA becomes a peak in 1 to 3 hours after partial hepatectomy but comes down to normal level in about 8 hours. Cycloheximide causes an overshooting of mRNA up to several hundred times in kidney, liver and spleen in 3 to 6 hours. Nuclear run-on assay revealed only a tenfold increase in c-myc transcription in regenerating liver. This discrepancy may be accounted for by the stabilization of mRNA by cycloheximide. Several deletion mutants of mRNA were constructed, and their stability tested in cos-cells. A region located near the 3’ end of mRNA was found to give strong influence on the stability of c-myc mRNA, The deletion at this region stabilizes the mRNA. He speculates that the region could possibly be recognized by RNase L.
Dr. Fujii-Kuriyama described recent progress in the study of cytochrome P-450 gene family. Comparison of gene structure of 38 different P-450 genes, including those for phenobarbital inducible, methylcholanthrene-inducible, C21 (steroid 21-hydroxylating) and SCC (steroid side chain cleavage) P-450’s, has led to a construction of a phylogenetic tree of these genes. For the MC-inducible P-450 gene, three regions of enhancer-like activity were identified by CAT assay of fusion genes; they span -3675 ~ -3067, -1683 ~ -1429 and ~ -1440 ~ -1029. Two types of core elements, designated XRE1And XRE2, were found in those regions. XRE’s show some homology with GRE (glucocorticoid responsive element) found in human metallothionein IIA, MMTV, MSV and chicken lysozyme genes. Gel-shift assays with a Hepa-1 cell extract using XRE2 as a probe identified a prominent band which could be competed by XRE2 sequence itself but not by nearby regions.
Dr. Darnell examined the expression of various liver-specific genes in different parts of the liver and found a position effect within a single cell lineage. For instance, certain P-450 and glutathione S-transferase species are expressed only near the central vein, sometimes along only one layer of the liver cell. This phenomenon may be explained by the effect of a ligand that bind specifically to the cell receptor and may constitute a basis of the “cell control” by the “gene control.” To approach these problems, Dr. Darnell isolated beta-IFN-inducible mRNA (pIF-1 and pIF-2) in the liver. These mRNA’s are induced in 10~30 minutes of exposure to beta-IFN and decreased in 6 hours. Gamma-IFN, cAMP, DMSO and Ca²⁺ ionophore do not induce them. A refractory period of 24~48 hours and superinduction by cycloheximide were observed. From genomic clones, a beta-IFN responsive region was identified at –73 ~ -122, where a gel-shift pattern was obtained after induction. He also examined 5’-flanking regions of mouse prealbumin gene, another liver specific gene, and found a potential enhancer region. This enhancer is tissue specific in that it works in HEPG₂ cells but not in HeLa cells. At present, however, no strict homology is identified among so-called liver specific genes.
On the whole, this seminar provided participants from both countries with an excellent presentation of new developments in each country and an exchange of new thoughts and ideas freely even before publication. As such the meeting was successful. The author was impressed to see that a new image was emerging in which multiple regulatory regions of a gene were using multiple transacting factors, and sometimes a region could bind with different factors. On the other hand, a factor could bind with different regulatory regions. Thus, the clarification of these networks of transcriptional regulation will be one of the main targets for future study.
As regards the relationship between the transcriptional control of oncogenes and other related genes, and malignant growth of the cell, much remains to be studied. However, fundamental knowledge on transcriptional control as discussed in this seminar will constitute a basis for its elucidation.


SEMINAR AGENDA AND PARTICIPANTS

(1) CELLULAR AND MOLECULAR MECHANISMS OF ONCOGENESIS AND CANCER IMMUNITY
Seattle, Washington, November 10-12, 1986

AGENDA

PARTICIPANTS

UNITED STATES

Dr. Jay Berzofsky
Metabolism Branch, NCI, NIH
Building 10, Room 4N115
Bethesda, MD 20892

Dr. Michael Brenner
Dana-Farber Cancer Institute
44 Binney Street
Boston, MA 02115

Dr. Robert Coffman
DNAX Research Institute
901 California Avenue
Palo Alto, CA 94304

Dr. Mark I. Greene
Department of Pathology & Laboratory Medicine
University of Pennsylvania
36th & Hamilton Walk
Philadelphia. PA 19104

Dr. Richard J. Hodes
Immunology Branch, NCI, NIH
Building 10, Room 4B17
Bethesda, MD 20892

Dr. Charles Janeway
Department of Pathology
Yale University Medical School
New Haven, CT 06510

Dr. Kathleen Kelly
Immunology Branch, NCI, NIH
Building 10, Room 4B47
Bethesda. MD 20892

Dr. Stanley J. Korsmeyer
Department of Medicine
Washington University School of Medicine
Howard Hughes Medical Institute
Box 8145
St. Louis, MO 63110

Dr. Dan Longo
NCI-FCRF
Building 567, Room 135
Frederick, MD

Dr. Alfred Singer
Immunology Branch, NCI, NIH
Building 10, Room 4B17
Bethesda, MD 20892

Dr. Philip W. Tucker
Department of Biophysics/Microbiology
University Science Center
University of Texas
5323 Harry Hines Boulevard
Dallas, TX 75235

Dr. Ellen S. Vitetta
Department of Microbiology
University of Texas
Southwestern Medical School
Dallas, TX 75236

JAPAN

Dr. Toshiyuki Hamaoka
Osaka University

Dr. Yoshiyuki Hashimoto
Tohoku University

Dr. M. Hosokawa
Hokkaido University

Dr. Katsuo Kumagai
Tohoku University

Dr. Nagahiro Minato
Jichi Medical College

Dr. Toshiaki Ohsawa
Tokyo University

Dr. Noriyuki Sato
Sapporo Medical College

Dr. Masaru Taniguchi
Chiba University

Dr. Junji Yodoi
Kyoto University

Dr. Tadamitsu Kishimoto
Osaka University

Dr. Toshio Hirano
Osaka University

Dr. Kazua Sugamura
Tohoku University

Dr. Kioshi Takatsu
Kumamoto University

Dr. Tadatsugi Taniguchi
Osaka University

Dr. Toshimitsu Uede
Sapporo Medical College

Dr. Takeshi Watanabe
Kyushu University



(2) CONFERENCE ON ‘REGULATORY ELEMENT IN NORMAL AND TRANSFORMED CELLS”

PARTICIPANTS

UNITED STATES

Dr. David Baltimore Whitehead Institute, MIT
Transcriptional control in lymphoid cells.

Dr. Michael Bishop
Hooper Institute University of California, San Francisco
Oncogene expression and function in Drosophila development.

Dr. James E. Darnell
Department of Molecular Cell Biology
Rockefeller University
Specific gene expression in hepatocytes and hepatoma cells.

Dr. Robert G. Roeder
Department of Biochemistry and Molecular Biology
Rockefeller University
Common and cell-specific factors active in in vitro transcription.

Dr. Robert Tjian
Department of Biochemistry
University of California, Berkeley
Interplay of multiple sequence specific factors.

Dr. Patrick H. O’Farrell
Department of Biochemistry
University of California, San Francisco
Role of engrailed locus in gene control in Drosophila.

JAPAN

Dr. Hideya Endo
Medical Institute of Bioregulation
Kyushu University
Conserved sequence specifically expressed in tumor cells.

Dr. Yoshiaki Fuji
Cancer Institute
Structure and regulation of multiple P-450 gene family.

Dr. Takeski Hayashi
National Cancer Center Research Institute
The Role of c-myc Gene in Carcinogenesis.

Dr. Tasuku Honjo
Kyoto University Faculty of Medicine
Aberrant expression of IL-2 receptor gene in adult T-cell leukemia cells.

Dr. Masami Muramatsu
University of Tokyo Faculty of Medicine
Structure and regulation of mammalian ribosomal RNA genes.

Dr. Yoshiaki Suzuki
National Institute for Basic Biology
Signals and factors for fibroin gene transcription.

Dr. Takeshi Watanabe
Institute of Bioregulation Kyushu University
Regulation of immunoglobulin heavy chain gene expression.