REPORTS ON SEMINARS

(1) Seminar on “Involvement of Growth Regulatory Genes in Cancer”
The seminar was held on February 15-17, 1992, at the Maui Prince Hotel, Maui, Hawaii. The organizers were Dr. Masaaki Terada (National Cancer Center Research Institute, Tokyo) and Dr. Stuart A. Aaronson (National Cancer Institute, Bethesda, Maryland). There were 14 participants, seven each from the United States and Japan and one observer from each county. Dr. Masaaki Terada welcomed the attendees and reviewed some of the exciting contemporary developments in the area of growth regulatory genes and the possibilities they present for cancer control.
The first report by Dr. Tony Hunter (The Salk Institute, La Jella, California) addressed itself to receptor protein-tyrosine kinases and phosphatases and their substrates. In previous studies Dr. Hunter’s group had identified two novel receptor-like protein-tyrosine kinases (PTKs). One of these, Eck, is mainly expressed in tissues containing growing epithelial cells, and is expressed on the basolateral surface of epithelial cell layers. Eck is a member of a small family of receptor PTKs, which includes Eph, Elk, Eed, Cek4/Mek4, Sek, Cek5/Nnk. Analysis of the 120-kDa Eck protein in immunoprecipitates from epithelial cells shows that Eck is a PTK. In an attempt to identify the Eck ligand, Dr. Hunter’s group found Eck in NIH/3T3 mouse fibroblasts and is now searching for tissue extracts or cell-conditioned media that can stimulate Eck tyrosine phosphorylation in these cell lines.
The second PTK, TrkB, isolated from a rat cerebellar library, was found to be closely related to but distinct from the Trk receptor-like PTK. TrkB is primarily expressed in brain, as a series of RNAs ranging from ~1-13 kb. Analysis of trkB cDNAs indicated that there are mRNAs encoding two different short forms of TrkB, which are both truncated just downstream of the transmembrane domain and have short, distinct C-termini. Using antisera to TrkB, Dr. Hunter identified the full-length protein as a 140-kDa glycoprotein. Based on the observation that NGF binds to and stimulates Trk PTK activity, it was shown that TrkB PTK activity is stimulated by NT-3 and BDNE, and the investigators are now searching for substrates for TrkB. In collaboration with Hakan Persson of the Karolinska Institute, Dr. Hunter has found that kindling-induced hipppcampal seizures induces rapid induction of trkB mRNA and TrkB protein in rat hippocampus. Concomitantly, BDNF mRNA is induced in the same areas of the hippocampus, suggesting that paracrine or autocrine stimulation is involved in repair of neuronal damage.
Dr. Hunter’s group has identified Tyr697, Tyr706, and Tyr721 in the kinase insert region of the CSF-1 receptor as autophosphorylation sites, and shown by mutagenesis that the phosphorylation of Tyr706 plays a role in induction of immediate early genes by CSF-1 and that Tyr721 acts as the phosphorylation site responsible for the binding of PI-3 kinase to the activated CSF-1 receptor. The mouse homologue of the PTP!!!receptor-like P. Tyr-phosphatase, PTP!!!was cloned. PTP!!!is phosphorylated constitutively in NIH/3T3 cells, predominantiy on a single serine residue, but a trace of phosphotyrosine is also detected. Dr. Hunter is currently testing whether its phosphorylation state and PTPase activity change upon mitogenic stimulation.
Searching for relevant substrates for PTKs, Dr. Hunter’ s group had previously shown that phospholipase C-!!!1 (PLC!!!l), the most abundant PLC in fibroblasts, is phosphorylated on tyrosine in PDGF- and EGF-treated cells, in conjunction with an increase in PLC activity. A small fraction of PLC!!!1 was found to be associated with proteins of 100 84 kDa but neither the stoichiometry of association nor the phosphorylation of these two proteins is affected by PDGF treatment. However these associated proteins may be important in PLC!!!1 regulation and the investigators have found that only two of the five available classes of anti-PLC!!!1 MAbs detect the complex. Using in vitro translation products of various regions of PLCl, Dr. Hunter has mapped the epitopes for all five classes of MAbs. The two classes of MAb that recognize the PLC1 complex bind to the SH2/SH3 regulatory region in the middle of the protein, and also recognize a cross-reacting 47-kDa protein, whose tyrosine phosphorylation is stimulated rapidly by PDGF treatment. In collaboration with Sue Goo Rhee (National Institutes of Health) and Yossi Schlessinger (New York University), Dr. Hunter’ s group has shown that this 47-kDa protein is the product of the nck gene, which was originally cloned by chance from macrophages. Like many of the receptor PTK substrates, Nck contains SH3 (three) and SH2 (one) domains, and it associates with the activated PDGF receptor via its SH2 domain. They have shown that Nck has multiple sites of serine phosphorylation, and that PDGF stimulates phosphorylation of a single tyrosine. Possible functions of Nck in receptor signalling are now being investigated.
Dr. Hunter’s group had previously identified ezrin (p81) as a substrate for a number of PTKs. Ezrin is related in its N-terminal 250 residues to the N-terminal 250 residues of the red cell protein band 4.1, talin, moesin, radixin, and two PTPs. In the red cell, band 4.1 acts as a linker between glycophorin in the plasma membrane and the actin/spectrin complex in the cortical skeleton. Dr. Hunter has identified Tyrl45 and Tyr353, which lie in the N-terminal domain and central!!!-helical regions of the protein respectively, as the two major tyrosine phosphorylation sites for the EGF receptor in ezrin. Tyrl45 is conserved moesin and radixin, two proteins closely related to ezrin, as well as in the two PTPs, suggesting that this might be a common tyrosine phosphorylation site. Dr. Hunter’s group has now mutated both sites to confirm their identification, and is expressing epitope-tagged forms of these mutants to determine the function of these phosphorylations. Dr. Masabumi Shibuya (Institute of Medical Science, University of Tokyo, Tokyo) presented two topics concerning receptor or receptor-like molecules. (1) Studies on activation of cellular genes coding membrane proteins, EGF-receptor, and CD43 in tumor cells. Gene amplification is one of the important mechanisms for activation of protooncogenes. However, the possibility of stuctural alternation of amplified genes and the molecular mechanism of the initiation of gene amplification have not yet been well characterized. Dr. Shibuya recently observed that human EGF receptor, c-erbB, gene is frequently activated in brain tumors stucturally and functionally. A deletion mutation within this gene starting from intron 1 to putative intron 7 took place in about 10% of glioblastoma multiforme, the most frequent and malignant human brain tumor, leading to constitutive activation of EGF receptor kinase. Sequence analysis of such mutated EGF receptor cDNA showed no mutation except for an 801 base pair deletion in the extracellular domain, indicating that a single deletion causes functional activation. This structural alteration of the EGF receptor was associated with gene amplification. The mutant gene was able to partially transform NIH/3T3 cells independently of the need for ligand, presumably because of constitutive kinase activation.
Dr. Shibuya also reported that about 20% of glioblastomas demonstrate gene amplification of wild-type EGF receptor. Overexpression of wild-type EGF receptor was able to transform NIH/3T3 only in the presence of high concentration of EGF. Thus, wild-type and mutated forms of EGF receptor are useful in studying the mechanisms involved in signal transduction and cell transformation.
(2) Friend erythroleukemia of the mouse induced by spleen focus-forming virus (SFFV) is one of the systems useful to characterize the cellular genes responsible for stepwise cell transformation. In order to obtain cellular sequences involved in transformation of erythrold cells, Dr. Shibuya attempted to isolate a new gene overexpressed in MEL cells using a differential hybridization method. He molecularly cloned a cDNA (melF) over-expressed in a murine Friend erythroleukemia cell line, F5-5. Southern blotting and sequence analysis revealed that the melF cDNA encodes a membrane protein of 395 amino acids that is the counterpart to human CD43 (leukosialin) surface antigen of lymphocytes in mice. The melF/mouse CD43 (mCD43) gene was found to be amplified 8- to 10-fold not only in F5-5 cells, but also in their sib cell lines, including the earliest clone established. This indicates that amplification of melF/mCD43 gene occurred at an early stage of malignant transformation of the F5-5 family. Furthermore, the same gene was found to be rearranged in another Friend erythroleukemia cell line, TSA8. In both cell lines F5-5 and TSA8, the levels of melF/mCD43 mRNA were 30- to 50-fold higher than that in uninfected spleen cells. Among normal tissues examined, the expression of melF/mCD43 gene was restricted to hematopoietic tissues. These results suggest that melE/mCD43 gene has an important role in progression of leukemic cells in the erythroid lineage and also has a physiological function in normal hematopoiesis.
Dr. Lewis Cantley (Tufts University School of Medicine, Boston. Massachusetts) described his studies on phosphatidylinositol 3-kinase and growth regulation. Phospbatidylinositol 3-kinase (PtdIns 3-kinase) was discovered because of its copurification with activated protein-tyrosine kinases of both the src family and receptor family. The lipid products of this enzyme, PtdIns-3,4P₂ and PtdIns-3,4,5-P₃ were not detectable in quiescent cells but were produced within minutes of cell stimulation by certain growth factors (e.g., PDGF, insulin, IGF-1) and found to be continuously elevated in cells transformed by certain oncogenes (v-src and polyoma middle t). These lipids are not in the canonical hatidylinositol turnover pathway and are not substrates for phospholipases C. PtdIns 3-kinase was purified to homogeneity from rat liver and shown to be a heterodimer of 85 kDa and 110 kDa subunits. The 85-kDa subunit had regions of homology to pp60^c-src (an SH3 domain and two SH2 domains) and a region of homology to BCR and the GTPase activating protein for rho (rho-GAP). The SH2 domains conferred binding of PtdIns 3-kinase to specific tyrosine-phosphorylated sequences found in protein-tyrosine kinases and targets of protein-tyrosine kinases. A serine kinase also copurified with PtdIns 3-kinase and quantitatively phosphorylated the p85 in the presence of Mn²⁺ and ATP. This phosphorylation inhibited the activity of the PtdIns 3-kinase. The mechanism of regulation of PtdIns 3-kinase by serine phosphorylation and by association with tyrosine-phosphorylated proteins was discussed.
Dr. Yoshiaki Ito (Institute for Virus Research, Kyoto University, Kyoto) discussed involvement of the polyomavirus enhancer binding protein 2 (PEBP2) in T cell-specific gene expression. PEBP2, also called PEA2, is composed of at least two subunits,!!!(30-35 kDa) and!!!(20-25 kDa). The!!!subunit alone can bind to DNA, but it does so more efficiently when it forms a heterodimer with the!!!subunit which, by itself, is unable to bind to DNA. The cDNA clones encoding the!!!and!!!subunits were cloned from the cDNA library prepared from RNA extracted from H-ras-transformed NIH/3T3 cells. Nucleotide sequence analysis of the!!!cDNA clones suggested that there were three subspecies encoding!!!l (22.0 kDa),!!!2 (21.5 kDa) and!!!3 (18.5 kDa) with common amino terminal portion but variable C-termini unique to each. Dr. Ito obtained evidence that these three subspecies are generated by alternative splicing.!!!l and!!!2 were able to form a heterodimer with the!!! subunit but!!!3 was not. The!!! subunit was expressed in all the cell lines tested, which included undifferentiated F9 cells, NIH/3T3 cells, and several B and T cell lines. The levels of expression were somewhat lower in F9 cells and higher in T cells, but the relative ratio of the three subspecies was nearly constant in all of the cell lines tested.
The major a mRNA species in H-ras-transformed NIH/3T3 cells was 6.5 kb. The cDNA clone, a10, corresponded to this mRNA and encoded a polypeptide of 56 kDa. When this cDNA was expressed either in E. coli or in COS cells, the primary translation product apparently degraded quickly and generated -30 kDa protein which bound to the PEBP2 binding motif. This stable fragment is likely to be an N-terminal half of the molecule.
Unlike the!!!subunit gene, expression of the!!!subunit gene was conspicuously cell type-specific.!!!10 mRNA was expressed in the T cell line, BW5147, as well as in NIH/3T3 cells, while it was undetectable in the B cell line, L1210, and F9 cells. Both BW5147 and L1210 contained a 3.6-kb RNA species which was undetectable in NIH/3T3 cells. The structure of the 3.6-kb species is currently unknown. This differential expression of mRNA strongly suggested that PEBP2 is involved in tissue-specific gene expression.
The “core” motif of MuLV enhancer is conserved among many strains of MuLV and is compatible with PEBP2 consensus sequence. Mutations in the core sequence of Moloney MuLV have been shown to drastically reduce T cell leukemogenicity, suggesting that the factor which recognizes the core motif is involved in T cell-specific gene expression. It was also noted that the enhancers of T cell antigen receptor (TCR)!!!,!!!,!!!and!!!contained the PEBP2 binding sites. In each of the enhancer elements, T!!!3 and T!!!4, or TCR!!!, a PEBP2 binding site was present. Together, T!!!3 and T!!!4 bore about 50% of T cell-specific function of the entire TCR!!!enhancer. Introduction of mutations in the PEBP2 site in T!!!4 did not significantly affect the activity, but those in T3 reduced the enhancer activity to 30% of the wild type level. Since the proto-oncogene product, Ets-l, also binds to T!!!3, the remaining activity was presumed to be due to Ets-1. Effects of mutations in the PEBP2 sites in TCR!!!and TCR!!!enhancers were also shown to be drastic.
When the CAT construct containing the T!!!3 and T!!!4 as enhancer elements was transfected into Ll210, the activity was very low. When the plasmid capable of expressing a10 mRNA was cotransfected into Ll210, dramatic increase in CAT activity was observed. These results suggest that PEBP2 plays a major role in T cell-specific gene expression. Studies to explore the possibility that PEBP2 is also involved in cell type-specific gene expression in other tissues and in stage-specific gene expression in developing mouse embryo are underway.
Dr. Michael Wigler (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) described conserved signal transduction pathways in yeasts and mammals. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have homologs of the mammalian RAS oncogenes. Dr. Wigler’s group has used these yeasts to study the RAS-dependent signal transduction pathways. In S. cerevisiae, RAS proteins regulate adenylyl cyclase, but have other functions besides. The RAS-effector pathways in S. pombe are less well understood. Genetic studies in S. cerevisiae indicate that the regulation of RAS is quite complex. Biochemical studies indicate that RAS-responsive adenylyl cyclase complex contains at least one other protein, called CAP. Genetic studies indicate that CAP is bifunctional. One function appears required for the proper regulation of adenylyl cyclase. S. cerevisiae CAP has a homolog in S. pombe, with similar function. Dr. Wigler and his associates utilized mammalian cDNA expression in yeast to characterize and isolate mammalian genes encoding proteins capable of interacting with, or substituting for, components of the RAS/adenylyl cyclase/CAP pathways in both yeasts. The nature of these genes was presented.
Dr. Kunitada Shimotohno (National Cancer Center Research Institute, Tokyo) presented his findings on hepatitis C virus and hepatocellular carcinoma. In Japan, patients with chronic non-A, non-B viral hepatitis are at high risk of developing hepatocellular carcinoma. Recently, hepatitis C virus (HCV) was shown to be the major causative agent of non-A, non-B chronic hepatitis, and analysis of its nucleotide sequence revealed the possibility that the virus represents a third member of the genus in Flaviviridae. The viral genome is variable and, based on the sequence variation, HCV can be classified into at least six types.
By using viral proteins expressed in E. coli for enzyme-linked immunosorbent assay, more than 85% and 95% of Japanese patients with non-A, non-B chronic hepatitis and hepatocellular carcinoma, respectively, were found to be HCV antibody-positive. Viral proteins were produced by processing of the primary precursor product which was translated from the largest open reading frame in the viral genome. The processing employs signalase which associates with the membrane of the microsome, and virally-coded proteinase.
The two regions in the putative envelope protein where nucleotide sequences show high diversity are known as hypervariable region 1 and 2 (HVR-1 and -2). Multiple HCV, differing in amino acid sequences in those regions, are often observed in the serum of the same patient. The significance of HV-1 and -2 to pathogenicity of this virus and the physiological role of viral replication remain to be clarified. However, such variation may represent a mechanism of virus propagation which eludes the host immunological surveillance system.
The mechanism involved in carcinogenesis by HCV infection, if any, remains to be clarified. Because of the lack of integration of HCV genome or gene into host chromosome, other mechanisms such as production of a viral oncogenic protein, disregulation of cellular gene expression by HCV infection or repeated wound-healing followed by genetic alteration of a host chromosome should be considered.
Dr. Shinichi Aizawa (The Institute of Physical and Chemical Research, Ibaragi) analyzed the multistep process of erythroleukemogenesis using mutant mice. In order to reveal cellular processes that are sensitive to F-SFFV-gp55 expression. Dr. Aizawa’s group had previously produced transgenic mice harboring the gp55 gene under the ubiquitous transcriptional regulatory unit of cyioplasmic !!! action gene. They showed that gp55 products uniquely penetrate erythroid precursor cells. The erythroleukemia development in these mice was under the control of the Fv-2 locus, suggesting the locus participates in the steps of signal transduction via gp55. Stochastic occurrence of the lenkemia even under Fv2^S/Fv2^S background, however, indicated that other events were required for the leukemia to develop in these mice; no chromosomal aberration was apparent in the erythroleukemia induced by gp55.
In an effort to delineate the events, Dr. Aizawa infected Moloney murine leukemia virus (Mo-MuLV) into the transgenic mice. The virus induced erythroleukemia within 50 days in all transgenic mice underFv2^S/Fv2^S background. It did not induce erythroleukemia in control nontransgenic mice or in transgenic mice under Fv2^r/Fv2^r background. Using the unique region of Mo-MuLV, common integration sites were identified among the leukemias induced. To date, five genomic DNA fragments flanking these common integration sites have been isolated. Three of these were the sites in Sfpi-1, pim-1 and p53 genes, respectively. In the remaining two cases, the isolated DNA fragments at integration sites did not reveal any transcripts in the erythroleukemia, and the identification of these genes is under examination.
The integration of Mo-MuLV in Sfpi-1 gene at its 5’ upstream sequences resulted in its activation. Then the retroviral vector carrying activated Sfpi-1 gene was infected into the gp55 transgenic mice. The virus dramatically accelerated the development of erythroleukemia, suggesting that activation of Sfpi-1 gene indeed cooperates with gp55 gene in erythroleukemia development. As a reference, a recombinant virus carrying the erythropoietin receptor was injected into the gp55 transgenic mice. Acceleration of erythroleukemia development was also apparent coinciding with the idea that gp55 activates erythroid progenitor cells by interacting with the erythropoietin receptor.
On the other hand, the integration of Mo-MuLV into the p53 gene disrupted the gene. To determine the cooperation of the p53-inactivation with gp55 gene in erythroleukemia development Dr. Aizawa attempted to produce mice deficient of the p53 gene. Homologous recombinant ES cells were efficiently obtained by use of negative selection with diphtheria toxin A fragment gene previously reported in the Proceedings of the Notional Academy USA on the c-fyn gene. The ES cells colonized into germ cells at high frequency. At the time of his presentation, the heterozygous mice were three months old but no occurrence of any tumors comparable to the human Li-Fraumeni syndrome had been observed. In addition, homozygous mice were live-born despite high p53 expression during organogenesis. Dr. Aizawa cultured neural precursor cells from neural tubes and cDNA corresponding to mRNAs from neural precursor cells were amplified by PCR using the sequences conserved among the tyrosine kinase family as primers. His group identified seven known genes, three possibly already identified genes and four unknown genes. He has developed a new method to isolate efficiently homologous recombinants of ES cells using diphtheria toxin A fragment gene as a negative selection against nonhomologous recombinants. They also developed a new strain of ES cells from F1 embryos between C57B1/6 and CBA mice. They have inserted LacZ into the third exon of c-fyn in ES cells, and studied the expression of c-fyn in embryos and mice by detecting LacZ products.
Dr. David Baltimore’s (Rockefeller University, New York, New York) presentation concerned making c-Abl an oncogene. c-Abl comes in two forms, called I and IV, that differ only in their N-termini. c-Abl(IV), the one his group has studied most, is nononcogenic even if overexpressed to the limit of toxicity, and is mainly nuclear. Dr. Baltimore’s laboratory has mutated c-Abl(IV) in various ways, and some mutants that revealed its oncogenic potential included deleting SH3, an F420V alteration and a replace of the N-terminus with a myristoylated gag segment. The oncogenic forms are all strictly cytoplasmic. By contrast to the SH3 deletion, altering SH2 usually counteracts oncogenicity.
SH2 acts to bind proteins that are tyrosine phosphorylated. A point mutant that abolished binding, abolished oncogenicity. SH3 binds a protein that Dr. Baltimore’s group has recently cloned. It is a GAP(rho)-related protein, but the SH3 binding region is not in an area of homology to any other protein. Finally, a model was presented at the meeting wherein the SH3 binding protein exhibited a negative role in transformation controlling both kinase activity and SH2’s ability to bind a phosphorylated target.
Dr. Masoaki Terada (National Cancer Center Research Institute, Tokyo) addressed his presentation to amplified genes in human cancer. One of the characteristics of cancer is aneuploidy, and increased content of DNA per cell is often associated with increased malignancy. Yet there has been only a limited number of known oncogenes amplified in human cancers. Genes which amplify frequently and give growth advantage to cancer are likely to be present. Using the gel DNA renaturation method, Dr. Terada was successful in isolating and identifying an amplified gene, designated K-sam, from a stomach cancer cell line, KATO III.
Sequence analysis predicted that K-sam encoded a membrane receptor with tyrosine kinase activity. K-sam was found to be relatively frequently amplified in poorly differentiated or diffuse types of stomach cancer, but not in the well-differentiated or intestinal types. In contrast c-erbB-2 was often amplified in the well-differentiated type and not in the poorly differentiated type. N-sam, a K-sam homologous gene, was isolated and found to be a human counterpart of the basic FGF receptor gene. K-sam is likely to be a gene for a receptor for growth fact(s) belonging to the heparin-binding growth factor family or FGF family. Dr. Terada found multiple forms of K-sam transcripts generated by alternative splicing. The presence of soluble forms of K-sam products was also predicted.
In esophageal cancer, HST1 and INT2 on 11ql3 were frequently amplified. By genomic walking Dr. Terada’s group identified two genes, EXPl and EXP2, on the same amplicon as HST1 and IN72 on 11ql3 with the total length of amplicon being approximately 150 kbp. The amount of EXPl and EXP2 mRNAs was increased in proportion to the degree of amplification, while mRNAs for HST1 and INT2 could not be detected even in the cells with amplification of the genes. It was found that the EXM2 cDNA sequence was identical to cyclin D cDNA. In one cancer, Dr. Terada also found many amplicons, which were isolated and identified by the genomic subtraction method.
Regulation of cell cycle progression by MyoD was presented by Dr. James Feramisco (University of California, San Diego, California). Skeletal muscle differentiation involves the removal of proliferating myoblasts from the cell cycle followed by fusion to form multinucleated cells and expression of a set of muscle-specific genes. A large body of work in recent years has established the importance of the myogenic determination proteins of the MyoD family in this process. MyoD is a basic helix-loop-helix (bHLH) protein which heterodimerizes with the ubiquitous bHLH proteins E12 and E47 to form anactive transcription factor complex which recognizes the “E box” (CANNTG) found in the promoters of many muscle genes.
In order to study the cell biology of MyoD in muscle differentiation, Dr. Feramisco’s group undertook a series of studies to investigate its role in the early stages of the differentiation process, the removal of cells from the cell cycle. They found that microinjection of pure, recombinant MyoD protein prevents serum-induced DNA synthesis--a result that is in agreement with previous expression studies from the laboratories of Drs. Phillipsen, Aaronson and Weintraub. By utilizing the microinjection approach, they were able to show that the inhibitory event occurs late in the G1 phase of the cell cycle. This late G1 timing led them to examine other events occurring at this point of the cell cycle. Accordingly, they have shown that during normal differentiation of musele cells, the tumor suppresssor protein RB becomes under-phosphorylated. They also found that this event takes place early in the differentiation process, before cell fusion or expression of muscle makers occurs. This was interesting because the unphosphorylated form of the RB protein is believed to be the active form which prevents entry into S phase. Dr. Feramisco’s laboratory has now established a link between the MyoD-induced arrest of cell cycle progression and RB by showing that microinjected MyoD protein prevents serum-induced phosphorylation of RB, as indicated by lack of extraction of RB from the nucleus. This result suggests that prevention of RB phosphorylation might be the mechanism through which MyoD is able to prevent entry into S phase. Further support for these ideas comes from studies using oncogenic Ras protein. This group has shown that microinjected, active Ras stops muscle cell differentiation and causes DNA synthesis to take place instead. In the muscle cell, this is correlated with phosphorylation of the RB protein. This was of interest because they have also been able to show that microinjection of high levels of Ras is able to overcome the cell cycle inhibitory effect of MyoD. Possible interactions between MyoD and RB are being examined.
A more detailed model where MyoD, in addition to its well established role as a transcription factor, also plays a part in the removal of myoblasts from the cell cycle can be proposed. Dr. Feramisco’s studies suggest that this function is mediated by a link between the MyoD protein and the RB tumor suppressor. This interaction results in the RB being maintained in the unphosphorylated, active form where the cells cannot enter S phase. This block in proliferation may aid in the progression-differentiation process.
Dr. Stuart Aaronson (National Cancer Institute, Bethesda, Maryland) described new approaches for isolation of growth regulatory genes. It has become increasingly evident that the pathologic expression of growth limiting genes in mitogenic signalling pathways plays an important role in the neoplastic process. The amplification and/or overexpression of genes encoding growth factors or their receptors contributes to a wide variety of human tumors. Other genes that act early in the intracellular transduction of growth factor signals have been implicated as oncogenes as well. Efforts to dissect the biochemical cascade initiated by growth factor triggering of receptors was discussed as well as new approaches aimed at identifying limiting genes in these pathways. Understanding gained from such approaches should be useful in efforts to develop new modalities of therapeutic intervention with cancer cells.
Dr. Yoichi Taya (National Cancer Center Research Institute, Tokyo) described protein kinases for cell cycle-dependent phosphorylation of the retinoblastoma (RB) protein. Growth suppressive activity of the RB proteins is believed to be inactivated by specific phosphorylation at G1/S border. Dr. Taya had previously reported that multiple sites of RB proteins are phosphorylated by cdc2 kinases purified from mouse FM3A cells. In follow-up, his group has been studying whether the odc2 kinases used are identical to the RB-kinase in vivo. For this purpose, they have highly purified the RB-kinase from FM3A cells and found that the major peak of RB-kinase activities can be separated from the major peak of histone H1 kinase activities by a Mono-S column chromatography. Moreover, cdk2, which was recently shown to be necessary for DNA replication. and cyclin A were detected in the same fractions by Western blotting. Surprisingly, c-myo protein was also detected in the same region.
In other experiments, Dr. Taya noted that RB proteins immunoprecipitated from human cell lysates are weakly phosphorylated in the absence of purified cdc2 kinase. Immunoblot analysis showed the presence of p34^cdc2 in the immunoprecipitates with anti-RB monoclonal antibody. In addition, the coprecipitated kinase was found to have the same substrate specificity as cdc2 kinase. The associated kinase activity was particularly high in cells arrested in G1/S and S phase by aphidicolin. Furthermore, RB proteins were shown to be phosphorylated in nuclear extracts by some endogenous cdc2-like kinase(s)
Studies on apoptosis mediated by cloned cDNA for cell surface antigen Fas were described by Dr. Shigekazu Nagata (Osaka Bioscience Institute, Osaka). Homeostasis in vertebrates is tightly regulated by cell death as well as by cell proliferation. The death of cells in embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, and normal tissue turnover is called “programmed cell death,” which is mediated by a process called “apoptosis.” Apoptosis can be distinguished from necrosis, which occurs as a result of injury, complement attack, severe hypoxia and hyperthermia. Morphological and biochemical analysis of apoptotic cell death indicated that apoptosis is accompanied by condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of nucleus, and extensive degradation of chromosomal DNA into oligomers of 180 bp.
Using human diploid fibroblast FS-7 cells as an immunogen, a monoclonal antibody (anti-Fas antibody) against human surface antigen was established. The Fas antigen is expressed in various human cells including myeloid cells, T lymphoblastoid cells and diploid fibroblasts. The Fas monoclonal antibody showed a cytolyic activity in cells expressing the Fas antigen especially in combination with IFN-!!!.
Complementary DNAs encoding the human and mouse Fas antigen were isolated from cDNA libraries of human T-cell lymphoma and mouse macrophage cell lines. The nucleotide sequence analysis of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319- (human) or 306- (mouse) amino acid polypeptide (Mr. 36,000) with a single transmembrane domain. The extracellular domainn consisting of about 150 amino acids, is rich in cysteine residue and shows a remarkable similarity to that of tumor necrosis factor receptors, nerve growth factor receptor, human B-cell antigen CD40, and rat T-cell antigen MRC OX40
Murine fibroblast L929 cells or T-cell lymphoma WR19L cells transformed with the human Fas antigen cDNA were killed by the anti-Fas monoclonal antibody. Morphological analysis of the dying cells by electron microscope indicated the Fas antigen can mediate apoptosis. Northern hybridization analysis of various mouse tissues indicated that the Fas antigen mRNA is expressed in the thymus, heart, liver and ovary but not in the brain, spleen and bone marrow. Treatment of various cell lines with interferon-Y significantly induced expression of Fas antigen mRNA in human Hr-29 colon carcinoma cells. The physiological function of the Fas antigen was discussed.
The scientific presentations closed with a presentation by Dr. John Isaacs (The Johns Hopkins Oncology Center, Baltimore, Maryland) who discussed the clinical implications of the regulation of programmed death of prostate and breast cancer cells. Although hormonal ablation is standard therapy for metastatic prostate and breast cancer, this therapy is rarely curative. The major reason for this is that metastatic cancer within an individual patient is heterogeneous, including both hormonal-dependent and -independent cancer cells. Thus, hormonal ablation docs not eliminate the hormonal-independent cancer cells within the patient. What is urgently needed is an effective therapy for hormonal-independent prostate and breast cancer cells which can be combined with any of the large variety of available forms of hormonal ablation to affect all of the heterogeneous tumor cell populations present within an individual patient with prostate or breast cancer.
Thus, new approaches to controlling hormonal-independent prostate and breast cancer cells are being sought. The growth of any cancer cell is determined by the relationship between its rate of proliferation and death. Only when the rate of cell death is greater than cell proliferation is the cancer cell eliminated. Thus, a successful treatment for hormonal-independent cancer cells can be obtained either by lowering the rate of proliferation and/or by raising the rate of cell death to a point where this exceeds the rate of cell proliferation.
Recent studies have demonstrated that androgen-dependent normal prostatic glandular cells, androgen-dependent prostatic cancer cells and estrogen-dependent breast cancer cells can be induced to undergo cell death following androgen ablation, and this death process docs not require the cells to be in the proliferative cell cycle. The death induced by androgen ablation in these nonproliferating cells occurs as an energy-dependent process collectively referred to as “programmed cell death” in which the cells actively commit “suicide.” Associated with the programmed cell death pathway is the enhanced expression of a series of genes (i.e., TRPM-2, TGF!!!1 c-fos, HSP-70, GRP-78, etc.), enhanced synthesis of a series of proteins (i.e., TGF!!!and EGF receptors, etc.), and the fragmentation of the genomic DNA into nucleosomal oligomers. This fragmentation of the genomic DNA is the irreversible commitment step in the death of the cell and is the result of the activation of Ca²⁺, Mg²⁺ -dependent endonuclease present within the cell nucleus. This activation is believed to be the result of a sustained elevation in intracellular free Ca²⁺ (Cai) induced following androgen ablation.
Additional studies have demonstrated that although hormonal ablation does not induce this programmed death pathway in hormonal-independent prostate and breast cancer cells these cells still retain the basic cellular machinery required to undergo such programmed death. This conclusion is based upon the observation that chemical agents high induce a “thymineless state” arrest the cells in the S phase of the cell cycle and activate this process, resulting in the programmed death of these cells.
There are a variety of antiproliferative chemotherapeutic agents which can induce this programmed cell pathway in sensitive target cells. Unfortunately, these agents usually lead to the death of cancer cells only during subsequent cell proliferation. Cancer cells not proliferating at the time of, or soon enough after, exposure are resistant to such cytotoxic agents since the cell has sufficient time to repair the damage induced before the next cell proliferation cycle. Unfortunately, the majority (i.e., 90%) of prostate and breast cancer cells within an individual patient are not actively proliferating and are thus resistant to standard cytotoxic chemotherapy. Therefore, what is needed is some type of cytotoxic therapy which induces the programmed death of hormonal-independent prostate and breast cancer cells without requiring the cells to proliferate.
Along these lines, it has been demonstrated that hormonally-independent prostate cancer cells can be induced to undergo the same programmed cell death pathway, if Cai is chronically elevated three- to sixfold for more than 12 hours by exposure to Ca²⁺ ionophores. Temporal analysis demonstrated that the death of these cells does not require cell proliferation and involves Ca²⁺ -induced fragmentation of genomic DNA into nucleosome-sized pieces as the commitment step in this process. These results demonstrate that even nonproliferating hormonal-independent prostatic cancer cells can be induced to undergo programmed cell death if a modest elevation in the Cai is sustained for a sufficient time. These observations identify Cai as a potential target for therapy for hormonal-independent prostate and breast cancer cells.
Dr. Aaronson thanked the participants for their contributions to a meeting characterized by impressive progress and new concepts. The material presented provided new insights into the basic genetic mechanisms of growth regulation, and new approaches which had immediate relevance to the basic cancer researcher as well as potential application at the clinical level.

(2) Seminar on Genomic Instability during Carcinogenesis and Tumor Progression
This seminar was held on February 15-17, 1992 at the Coco Palms Resort, Kauai, Hawaii. The organizers were Dr. Curtis C. Harris, National Cancer Institute, Bethesda, Maryland and Dr. Setsuo Hirohashi, National Cancer Center Research Institute, Tokyo. There were 14 participants: seven from Japan and seven from the United States.
Dr. Harris welcomed the attendees and introduced Dr. Takashi Sugimura (National Cancer Center), Program Coordinator of the Etiology Program Area, U.S.-Japan Cooperative Cancer Research Program, who delivered the introductory remarks, presenting the brief history of the U.S.-Japan Cooperative Research Program, and the reason why the topic of this seminar was selected.
Recent remarkable progress in molecular genetics in cancer research has made it clear that carcinogenesis and tumor progression are caused by accumulation of multiple chromosomal alterations and mutations in proto-oncogenes and tumor suppressor genes. The aim of this seminar was to discuss the most recent knowledge on the molecular mechanisms leading to genetic alterations during the course of carcinogenesis and tumor progression. It is strongly suggested that genomic instability is one underlying mechanism for the accumulation. Leading scientists in the fields of cell cycle control, mitosis, recombination. mutation and gene amplification were invited to this seminar.
The current knowledge on the biology of the mitotic apparatus was reviewed by Dr. Bill R. Brinkley (Baylor College of Medicine, Houston, Texas) and a molecular scheme of the centromere-kinetochore complex in animal cells was presented. Using sera from patients of autoimmune disorder CREST syndrome, 50-60% of which contain anti-centromere antibodies, a family of centromeric proteins has been identified. Among them, CENP-B, an SO-kD acidic protein with DNA-binding property, was demonstrated to bind with a 17 base pair-DNA motif which is located within a centromeric repetitive DNA sequence of each chromatid. CENP-B also binds with certain microtubule-associated proteins (MAPS), and as a result CENP-B and MAPS make a cross-bridge between the centromere of a metaphase chromosome and mitotic spindle microtubules. Progress in characterizing the function of centromeric proteins was also presented. He also discussed the mechanism of asbestos-induced anueploidy in human cells. Asbestos fibers, endocytosed by cultured human lung cells, caused a precipitous increase in mitotic spindle aberrations, micronuclei and multinucleated cells within two to five days after exposure. More significantly, when crocidylite and crysotile fibers are incubated with whole-cell homogenates from the same cells, selective binding of several important spindle proteins, including tubulin, actin and centromeric proteins, were demonstrated by SDSPAGE and Western blotting. The selective affinity of asbestos fibers for mitotic spindle proteins could account for their targeting to the mitotic apparatus and induction of aneuploidy leading to the development of malignant human mesotheliomas.
Dr. Hiroto Okayama (Research Institute for Microbial Diseases, Osaka University, Osaka) discussed dysregulation of human mitotic control genes in cancer cells and its implication for their genetic instability. The G2 phase of the cell cycle is the period in which entry into mitosis is regulated by a complex system involving cdc2⁺ protein kinase as a key controller. In fission yeast the activity of cdc2⁺ kinase is regulated by the cdc25⁺ phosphotyrosine phosphatase and weel⁺/mik1⁺ tyrosine kinases, and aberrant expression of these mitotic control genes, such as overexpression of edc25⁺ combined with underexpression of weel⁺, disturbs coordinated cell division and prematurely induces mitosis, resulting in the generation of anucleate and aneuploid cells.
This regulatory system is evolutionarily well conserved throughout higher eukaryotes. String is a Drosophila homolog of cdc25⁺ and CDC25Hs (CDC25HuA) is a human homolog isolated by Sadhu, K., et al. (Proc. Natl. Acad. Sci. USA, 87; 5139-5143, 1990). Recently a weel⁺ homolog (WEE1Hu) and two additional cdc25⁺ homologs [CDC25HuB and CDC25HuC] from human cells were cloned in Dr. Okayama’ s laboratory. CDC25HuA and CDC25HuB are predominantly expressed in mammalian cells during G2, sharing remarkable similarity with the cde25⁺ gene in yeast. In most of the cells examined, CDC25HtiB is expressed 10- to 100-fold more than CDC25HuA The mRNA steady state of CDC25HuB was particularly high in various cancer cells and SV40-transformed cells, with a noticeable correlation between the extent of aneuploidy and the level of its expression. Moreover, the expression of CDC25HuA, CDC25HuB and cdc2 was no longer cell cycle-dependent in the SV40-transforrned cells, and the aberrant expression of these mitotic control genes occurred immediately after transformation of human fibroblasts by SV40.
Dr. Takeharu Nishimoto (Graduate School of Medical Science, Kyushu University, Fukuoka) discussed the RCC1 gene which was isolated by complementing the tsBN2 mutation which is a temperature sensitive mutation of the BHK21 hamster cell line. The RCC1 gene encodes a 45 kD nuclear DNA-binding protein and appears to be essential in sequential coupling DNA replication and mitosis.
Upon loss of RCC protein, tsBN2 cells which are replicating enter mitotic phase. Recently, Bischoff, F.R. and Ponstingl, H. (Nature, 354; 80-82, 1991) found that RCC1 protein binds to the small G protein, i.e., Ran, and has a catalytic activity of guanine nucleotide exchange on this protein. Thus, the RCC1/Ran system functions as a member of GTP cycle in the nuclei. Dr. Nirshimoto and his co-workers proposed that the DNA binding activity of RCC1 took a role in the signal transduction from DNA to the regulator of MPF (maturation promoting factor). However, the RCC1 protein without DNA binding domain complemented tsBN2 mutation efficiently and the endogenous RRC1 disappeared in these ts⁺ transformants of tsBN2 cells transfected with the deleted RCC1 CDNA at 39.5 C. Although the majority of the deleted RCC1 protein was in the cytoplasm, a significant amount of the deleted one was kept in the nuclei and co-migrated with the nucleosome-fraction in sucrose-density gradient analysis. Since the deleted RCC1 protein did not bind DNA molecule by itself, these results indicated that RCC1 protein binds to the chromatin with the aid of other unknown protein(s).
Since the premature chromosome condensation should cause chromosomal loss, the defect of RCC1/Ran system may promote carcinogenesis. It is an interesting question to investigate the RCC1/Ran system in cancer cells.
Human cancers such as colorectal carcinomas are known to exhibit reduction of telomeric sequences. A chromosome with such terminal deletion tends to generate a reactive end that may lead to additional chromosomal aberrations upon cell division. These aberrations are a characteristic of cancer cells, and telomere reduction is thought to contribute to carcinogenesis and subsequent tumor progression.
Dr. Ryo Kominami (Niigata University, School of Medicine, Niigata) reported that in addition to the reduction of telomeres, size-variants are frequently observed in tumors, subclones of tumor lines and an established murine cell line. This indicates that telomeres are highly unstable in both tumors and certain cultured cell lines that are free from the regulation of living organisms. Since the changes were detected as discrete bands, they were probably caused by recombination between telomeres. He also described conditions in which telomere elongation could also be made by the de novo synthesis of a telomere by the ribonucleoprotein enzyme telomerase.
Dr. Gregg B. Morin (University of California Davis, California) summarized DNA recognition properties of human telomerase and the role in carcinogenesis. Human telomerase, in vitro, recognizes the trancated chromosome end and adds telomeric repeats. The DNA sequence requirements for telomerase recognition of the DNA primer are very minimal. Since telomerase may not be continuously expressed in somatic cells, it must be activated to contribute to carcinogenesis. The importance of telomere changes in chromosome instability has been appreciated but its exact mechanistic role still remains unclear.
Patterns of spontaneous mutations in experimentally induced conditions and patterns of mutations in cancers were compared by Dr. Lawrence A. Loeb (Gottstein Memorial Laboratory, University of Washington, Seattle, Washington). Evidence indicates that the point mutation frequency in somatic cells is 1.4 x 10-10 mutations/nucleotide/cell generation. Three potential sources of spontaneous mutations, 1) chemical hydrolysis of the glycosylic bond in DNA; 2) damage to DNA by oxygen free radicals; and 3) misincorporations by DNA polymerases, were analyzed. Each process, if not repaired, could generate about 3000 mutations/cell/day. Spontaneous chemical hydrolysis of DNA generates abasic sites that direct the preferential incorporation of deoxyadenosine. Replication of DNA damaged by oxygen free radicals results in a spectrum of nonrandom singlebase mutations, most frequently C-> T and G-> T substitutions. In addition, an unusual mutation, two adjacent thymidines substituting for two cytidines, is produced by either oxygen free radicals or UV irradiation. Infidelity of different DNA prlymerases yields a characteristic pattern of single base substitutions, predominantly transitions. A comparison of these data with the spectrum of spontaneous mutations that have been analyzed in different laboratories indicates that abasic sites are not a major premutagenic event and favor replication errors or oxygen free radical damage as major sources of spontaneous mutations.
Dr. Hiroyasu Esumi (National Cancer Center Research Institute, Tokyo) reported an unusual finding on altered specificity of mRNA splicing in analbuminemic rat liver during carcinogenesis and aging. In analbuminemic rats, a seven-base-pair deletion in the splice-donor site of the 9th intron of albumin gene blocks normal albumin mRNA splicing resulting in a production of 9th-exon-skipped albumin mRNA. Only a trace amount of albumin synthesis is detected in the liver of analbuminemic rats and few hepatocytes are positive for albumin immunohistochemically at 4 weeks after birth. However, a large number of hepatocytes become albumin-positive after administration of hepatocarcinogens such as 3’-MeDAB, Glu-P-1 and AAF or during aging. Western blot analysis of the liver of aged or carcinogen-treated analbuminemic rats showed that small molecular weight anti-albumin reactive proteins which are not present in the younger untreated rat liver are present. Subcellular localization of the albumin-related proteins was found to be a particulate fraction in which albumin is not found normally. Northern blot analysis revealed that small sized mRNA increased during the course of caricinogenesis and aging and those mRNAs were found to be 9th and 10th exons-skipped mRNA. The fate of albumin-related protein translated from aberrantly spliced mRNA was examined by expressing these mRNAs in various hepatoma cells. The results showed that truncated albumin due to the 9th and 10th exon deletion accumulated in the particulate fraction of the cells. He concluded that alteration of the specificity in mRNA splice site selection resulted in an ectopic accumulation of aberrantly synthesized protein. Not only the structural stability of the gene but also functional stability of RNA processing is important in changes in phenotypic expression during carcinogenesis.
Gene amplification is frequently observed in tumors and transformed cell lines, and is one marker for genomic instability. Dr. Thea D. Tlsty (University of North Carolina, Chapel Hill, North Carolina) reported a correlation between the ability of cells to amplify endogenous genes and their ability to form a tumor when injected in the appropriate animal. Human cells were exposed to N-(phosphonoacetyl)-L-aspartate (PALA), which specifically inhibits aspartate transcarbamylase activity of the CAD enzyme, and PALA-resistant subclones were selected. She observed a striking parallel between the ability of these cell lines to become resistant and the ability of the same cell lines to form tumors after injection into day-old syngeneic rats Molecular analyses of PALA-resistant subclones revealed that this resistance was due to amplification of the CAD gene. Tumorigenic cell lines demonstrate a frequency of gene amplification that can be greater than 100 times that seen in non-tumorigenic cell lines. In contrast, gene amplification is undetectable in primary, diploid cell populations. These results demonstrated that a dramatic difference exists between primary diploid cell populations and immortalized populations in their ability to amplify genomic sequences and suggested a significant difference in genetic instability between these two cell types. The approach using somatic cell hybrid to identify the gene(s) controlling the susceptibility for gene amplification was also discussed.
The genes that encode the variable regions of immunoglobulins and T-cell receptors are assembled by a common recombination system referred to as VDJ recombination÷ which are likely controlled by the RAG-1 and RAG-2 genes. Frederick W. Alt (Howard Hughes Medical Institute, Columbia University, New York, New York) has investigated the function of the RAG-2 gene by constructing RAG2 deficient transgenic mice. Such mice are viable but show a severe combined immune deficiency (SCID) due to a total inability to initiate VDJ recombination. RAG-2 deficient mice have no obvious defect in any tissue or lineage other than lymphocytes; thus, they concluded that VDJ recombinase activity and RAG-2 gene function is required only for lymphocyte development. Since homozygous SCID mutants also have a defect in the general DNA repair processes, he is currently isolating genes which complement the defects of both DNA repair and VDJ recombination. Such genes are likely to be candidates of genes for genomic instability in general.
Dr. Curtis C. Harris (National Cancer Institute, Bethesda, Maryland) reviewed involvement of p53 tumor suppressor gene in human carcinogenesis. Mutations in the evolutionarily conserved codons of the p53 tumor suppressor gene are common in diverse types of human cancer. The p53 mutational spectrum differs among cancers of the colon, lung esophagus, breast, liver, brain, reticuloendothelial tissues and hemopoietic tissues. Analysis of these mutations can provide clues to the etiology of these diverse tumors and to the function of specific regions of p53. Transitions predominate in colon, brain and lymphoid malignancies, whereas G:C to T:A transversions are the most frequent substitutions observed in cancers of the lung breast and liver. Mutations at A:T base pairs are seen more frequently in esophageal than in other solid tumors. Most mutations in lung, breast and esophageal carcinomas are dispersed among numerous evolutionarily conserved codons. In liver tumors in persons from geographic areas in which aflatoxin B1 and hepatitis B virus are cancer risk factors, most mutations are at one nucleotide pair of codon 249. One hypothesis is that aflatoxin B1 is metabolically activated to form the promutagenic N7dG adduct and enhanced cell proliferation due to chronic active viral hepatitis allows both fixation of the G to T transversion in codon 249 of the p53 gene and selective clonal expansion of the cells containing this mutant p53 gene. Induction of skin carcinoma by ultraviolet light is indicated by the occurrence of pS3 mutations at dipyrimidine sites including CC to TT double base changes. Therefore, these differences in p53 mutational spectra among human cancer types reflect the etiological contributions of both exogenous and endogenous factors to human carcinogenesis, and the selective clonal expansion advantages of various mutant p53 genes in different human cell types.
Dr. Jun Yokoto (National Cancer Center Research Institute, Tokyo) described the presence of multiple genetic alterations in human lung and colorectal carcinomas. The purpose of this study was to clarify the molecular mechanisms of tumor progression. In lung carcinoma, inactivation of the RB and p53 genes as well as allelic losses on chromosome 3p were detected frequently in primary tumors. In addition to these genetic alterations, accumulation of chromosome 1 Ipallelic deletions and myc amplification in the process of metastasis were observed in some cases. In colorectal carcinoma, alterations of the p53 and DCC genes were detected in approximately 70%, of primary tumors and in 100% of liver metastases. Furthermore, allelic losses on chromosomos 13q, 14q and ISq accumulated during tumor progression. Among several genetic alterations, p53 alterations seem to occur relatively early in tumor progression and may be critical events for the malignant conversion of premalignant cells. The results suggest that at least three to five genes are mutated in tumor cells with metastatic ability, and that the p53 gene would be a trigger for genomic instability occurring in vivo during tumor progression.
SV40 T antigen binds, thereby inactivating p53, and it is well known that cultured SV40 transformed cells develop marked chromosomal aberrations. Dr. Michael A. Tainsky (University of Texas, M. D. Anderson Cancer Center, Houston, Texas) reported that fibroblasts from patients with Li-Fraumeni syndrome, which possessed germline mutations of p53, acquired an altered morphology, chromosomal anomalies and the ability to exhibit anchorage-independent growth as they were cultured in vitro. Unlike normal human fibroblasts, these cells became continuous cell lines. These findings may suggest that p53 might be involved in the regulation of the expression of various mitotic control genes, and that inactivation of p53 by mutations might result in their aberrant expressions, thereby inducing genetic instability.
Dr. Setsuo Hirohashi (National Cancer Center Research Institute, Tokyo) presented data on alterations of tumor DNA ploidy and tumor suppressor genes in human cancer progressron.
Inactivation of multiple tumor suppressor genes plays an important role in tumor progression and, directly or indirectly, causes chromosomal and genetic instability that leads to the emergence of more malignant clones. Hepatocellular carcinomas (HCCs), in which morphological evidences of stepwise progression are clearly observed, were selected for study. In liver damaged by virus infection, small nodular lesions develop as a clonal expansion of hepatocytes. Within these nodules, HCCs of the moderately or poorly differentiated type often emerge as a subclonal expansion and form nodule-in-nodule lesions. The ordinary HCCs formed by progression show highly increased cell proliferation, increased number of nuclear organizing regions (NORs) and aneuploidy in 'some tumors, together with inactivation of multiple tumor suppressor genes including p53 and RB. Association between anueploidy and p53 mutation was observed not only in BCC but also in other cancers such as gastric cancer and ovarian cancer. Furthermore, Dr. Yoshinori Murakami (National Cancer Center Research Institute, Tokyo), who attended as an observer, added data of a close association of the presence of p53 mutation with chromosomal aberrations in cultured human carcinoma cell lines.
Thus, the most recent information on a) genetic and chromosomal alterations in cancer, b) their role in carcinogenesis and tumor progression c) role of genomic instability in accumulating such alterations, and d) fundamental mechanisms possibly involved in genomic instability were exchanged. Although this is a relatively new field in cancer research, the participants predicted that there will be rapid progress in the near future disclosing the mechanisms of genomic instability occurring in cancers and their relation to the multiple genetic and chromosomal alterations observed during carcinogenesis.

(3) Seminar on “Transgenic Animals in Cancer Research”
This seminar was held on February 23-26, 1992 at the Oiso Prince Hotel, Kanagawa, Japan. The organizers were Dr. Motoyo Katsuki, Kyushu University, Medical Institute of Bioregulation, Fukuoka, Japan and Dr. George F. Vande Woude, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland.
There were seven speakers from Japan, five speakers from the United States and also four observers from Japan. The purpose of the meeting was to discuss and exchange information on the use and knowledge of transgenic animals particularly for purposes of studying the process of oncogenesis and proto-oncogene function. The opening remarks by Drs. Katsuki and Vande Woude covered the importance of transgenic animal studies in understanding oncogene function, but also emphasized the importance of the mouse system as a model for studying human diseases.
The four-session program with three speakers per session began with the characterization of mouse embryonic stem (ES) cell system. The first speaker, Dr. Colin L. Stewart (Roche Institute of Molecular Biology, Nutley, New Jersey) presented his studies on the function of the embryonic cytokine, LIF, in development. LIF has many different functions as a cytokine; among them, is the inhibition of differentiation of mouse embryonic stem cells. Dr. Stewart and his colleagues have examined LIF expression during early development in mice. They found that maximal expression occurs in the uterus during the fourth day of pregnancy and coincides with the time blastocysts are present. They have also been able to show that LIF is under maternal control since pseudopregnant females also express the cytokine and expression always precedes implantation. Their studies suggest that the principle function of LIF in vivo is to regulate the growth and initiation of blastocyst implantation. Dr. Stewart has mutated the LIF gene by using homologous recombination in ES cells and they have been able to introduce the mutated gene into the germ line of mice. Mice homozygous for LIF deficiency are viable although they, as adults, only attain 60-70% the size of their wild type or heterozygous siblings. Homozygous males are fertile, but homozygous females are sterile and do not produce any offspring. The sterile females produce viable blastocysts but the blastocysts do not implant thus providing additional evidence that the burst of LIF expression that occurs in the fourth day of pregnancy is required for implantation. Since adult mice deficient in LIF are viable and embryos deficient in LIF are obtainable, future studies will center on analyzing the in vivo requirement of LIF for generating teratocarcinomas in adult mice and also, perhaps, in sustaining the growth of other types of tumors.
Dr. Shin-ichi Aizawa (The Institute of Physical and Chemical Research, Koyadai, Tsukuba. Japan) presented his work on the improved mouse’s ES cell technology and gene targeting work on the c-fvn oncogene. His method of negative selection using the diphtheria toxin A fragment (DT-A) and his high frequency of germ line transmission was very impressive.
Dr. Kirk Thomas (Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah) was the last speaker of the session. He discussed the results of the extensive analysis of the requirements of efficient homologous recombination in murine embryo-derived stem cells used to generate germ line gene knock-outs. They find that efficient recombination is a function of both the length and extent of homology between the targeting vector and its genomic target but that there are unexplained differences in recombination efficiencies between different genetic loci.
They have used this technology to target null mutations to the wnt-1 (int-1) proto-oncogene. Although identified because of its involvement in virally-induced mouse mammary carcinomas, the wnt-1 gene was known to be expressed primarily in the developing nervous system of mid-gestation mouse embryos. Consistent with this expression pattern, it was shown that animals homozygous for the targeted mutations in wnt-1 have several nervous system abnormalities which are first detected at embryonic day 10 as a reduction in neural tissue at the midbrain-hindbrain junction. At birth this defect is translated into a marked reduction in the amount of both cerebellar and midbrain tissue. They observed a striking variability in expressivity of the wnt-1 mutation manifest both in the life span of homozygous individuals and in the quantity of neutral tissue at a particular stage of development. The life span ranged from death in utero to survival to adulthood with severe ataxia. The neural deficiencies ranged from the absence of the anterior cerebellar folia to the complete absence of the cerebellum as well as of adjacent midbrain nuclei. Although the tissues expressing the highest levels of wnt-1 mRNA, other regions of the nervous system known to express the wnt- I gene were seemingly unperturbed by the mutation. It was hypothesized that other genes in the wnt-gene family may substitute for wnt-1 in mutant animals.
The first two speakers of the second session, Dr. Luis F. Parada (ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland) and Dr. Hisato Kondoh (Nagoya University, Nagoya. Japan) focused on N-myc proto-oncogene germ line targeting studies. The N-myc gene has been implicated in cancers of embryonic (Wilm’s tumor, retinoblastoma) or of neuroendocrine (neuroblastoma, SCLC) origin. While, as expected, c-myc is expressed in a broad spectrum of tissues, N-myc expression was found primarily in embryonic tissues and specifically in epithelial tissues including neuroectoderm and in neural crest derivatives. Dr. Parada first described the expression of N-myc in normal tissues during development where he compared the expression of N-myc to that of the closely related c-myc proto-oncogene. These studies have shown that N-myc and c-myc are rarely co-expressed in the same cell types and indicate a correlation between fetal N-myc expression (kidney, retina, PNS, lung epithelium) and N-myc-associated tumors. The aim of these studies was to generate mouse models to assess N-myc function during development.
Both investigators reported that mice homozygous for the mutated N-myc allele do not develop to term. Analysis of the mutants during embryogenesis indicate that the mutants arrest during organogenesis. The mutant embryos exhibit disorganized epithelial and neural crest derivatives. The observed defects are thus consistent with the observed patterns of expression in normal embryos indicating that N-myc expression is required for the correct development of these tissues. The N-myc mice will be extremely valuable in dissecting N-myc function and the molecular basis of N-myc participation in malignancy.
The third speaker of the second session was Dr. Ken-ichi Yamamura (Kumamoto University, Kumamoto, Japan). In the first part of his presentation he described his transgenic mouse studies using the rearranged or activated Eµdryc oncogene locus. His studies brought out the importance of the effects of genetic background and polygenic effects on the tumorigenic phenotype. The second half of his talk dealt with a system he has developed for trapping genes involved in embryonic development.
The third session topics dealt with transgenic studies using transgenes that are directly associated with carcinogenesis. The first speaker, Dr. Kiyoji Tanaka (Osaka University, Osaka, Japan), described the use of a human cDNA of the DNA repair enzyme responsible for xeroderma pignentosum as a transgene.
Dr. George Vande Woude (ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland) presented the studies from his laboratory describing transgenic mos mice. Four transgenic strains of mice were generated containing a retroviral long terminal repeat linked to the murine c-mos proto-oncogene coding sequences. The first mos dependent transgene phenotype observed in these animals was a dominant change in secondary lens fiber epithelial cell differentiation that led to perforation of the embryonic membrane on the posterior part of the lens capsule. No hyperplasia or neoplasia, however, was observed in the lens. These mos transgenic animals also displayed severe behavioral abnormalities including circling, head-bobbing and head-tilting. Pathological examination showed that they all exhibited neuronal and axonal degeneration, as well as gliosis and the severe neuropathological alterations were directly correlated with the expression of the mos transgene. In spite of this severe neuropathological phenotype, neoplastic disease was not observed in the brain and the animals lived for periods of 12-18 months. Mice in three of the four mos transgenic lines did, however, develop frequent pheochromocytomas and/or medullary thyroid C-cell carcinomas after latent periods of eight months.
In humans, pheochromocytomas and medullary thyroid carcinomas are diseases associated with the autosomal dominant neoplastic syndrome, multiple endocrine neoplasia type 2 (MEN 2). The tumors originate as small nodules of hyperplastic cells in the adrenal medulla or as C-cell hyperplasia of the thyroid medulla. Both types are derivod from neural crest cells; they also occur bilaterally with multifocal lesions, indicative of an inherited genetic disorder.
Three of the four mouse mos transgenic lines displayed tumors remarkably similar to MEN 2 syndrome and greater than 60% of these animals develop pheochromocytomas or medullary thyroid neoplasms. Moreover, the tumor histology, pattern of presentation, and the staining pattern with regard to neuron-specific enolase in pheochromocytomas or calcitonin in medullary C-cell neoplastic disease was indistinguishable from the MEN 2 syndrome. The type of tumor varied in a line-dependent manner: thus, one line produced predominantiy pheochromocytomas, a second line produced –63% medullary C-cell carcinomas, and a third line produced both kinds of tumors. A fourth line, displaying the mos transgene eye defect and neuropathological changes did not develop tumors. However, when this transgenic line was crossed onto a different mouse background, the FI progeny developed typical MEN 2 syndrome tumors. Dr. Vande Woude suggested that the background and/or integration site influences the specific tumor type. As with the E-myc tumorigenesis described by Dr. Yamamura in the second session, the MEN 2 phenotype was also dependent on the genetic background of the canier mice, suggesting the importance of polygenic interaction for tumorigenesis. A possible explanation for the mos transgene tumor specificity may be related to the normal proto-oncogene expression in the brain. Thus, the neural crest origin of the tumor cells might suggest that a specific target is present in this tissue. This would also be present in cells of the brain and might explain the severe neuropathological changes in these mice. They have shown that tubulin is a potential target of mos protein and overexpression of the proto-oncogene product in cells of neural origin could markedly affect microtubules. Moreover, the long latent period suggests a genetic instability mechanism and this would be consistent with the models proposed for how mos could promote genetic instability.
Dr. Motoya Katsuki (Kyushu University, Fukuoka, Japan) presented the dramatic effect of mouse strains carrying the human proto-oncogene, c-Ha-ras, on in vivo tumorigenesis. They observed astonishingly high tissue-specific tumor formation associated that correlated with somatic mutations in the c-HA-ras gene. Dr. Katsuki showed how this transgene strain is useful for studying the effects of chemical carcinogens and anticarcinogens.
In the last session, Dr. Stephen Hughes (ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland) presented studies from his laboratory on transgenic mice carrying the ski oncogene. They find that ski gives rise to a very unusual growth of the skeletal musculature. In the initial series of experiments, they derived three independent lines of mice that share a distinctive phenotype: they have abnormally large (two or three times normal) skeletal muscles, but virtually no fat. All three lines of mice express the transgene at high levels in skeletal muscles, but not in other tissues or organs tested. This remains a puzzling observation. The Moloney sarcoma virus LTR, which was used as a promoter in these experiments, has been linked to other genes and used to make lines of transgenic mice that do not show selective expression in muscle. In collaboration with Dr. Alan Kelly’s laboratory (University of Pennsylvania) they have examined one of the three lines of muscular mice in more de tail. The primary effect is hypertrophy, a significant increase in the size of the individual muscle fibers without au increase in the number of fibers. Most of the skeletal muscles are involved, with the exception of the soleus, the diaphragm, and the tongue. The level of the ski transgene expression is much lower in the diaphragn and the soleus than in the other leg muscles, which show significant hypertrophy. Mammalian muscles are composed of several different types of fibers. In the one line of mice that has been studied in detail, specific types of muscle fibers are affected by ski. The different fiber types contain unique myosin heavy chains (MHC) that can be identified with specific monoclonal antisera. This technique was used with sections made from muscle that had undergone ski-induced hypertrophy. Only type II fast fibers undergo hypertrophy; both type IIb and IIx fibers appear to be affected. They have also generated transgenic pigs and these animals also show some of the same increase in skeletal muscle and decrease in body fat
The second speaker of the last session was Dr. Yuichi Obata (Aichi Cancer Center Research Institute, Nagoya. Japan). He described investigations in the development of leukemia and immunology using the mouse thymus leukemia (TL) antigen gene. The expression of TL antigen is dependent on the strain of mouse: e.g., Balb/c mice express TL antigen in the normal thymus, but C3H/HE do not. T-cell lymphomas induced by X-ray irradiation result in TL antigen expression in C3H/HE mice.
The last presentation was by Dr. Toshihiko Shiroishi (National Institute of Genetics, Mishima, Japan). He presented his studies on the genetic recombination and has been studying recombination “hot spots” near or within the H-2 locus (major histocompatibility complex; MHC) of the mouse. He finds a high frequency of spontaneous mutations in the mouse genome. The mechanism of the mutagenesis is not known at all. All participants agreed that the conventional transgenic animal technology as well as gene targeting techniques will be very powerful tools to cancer research and basic science. Both organizers, Drs. Katsuki and Vande Woude, concluded that the best reward of the meeting was the development of a stronger friendship between Japanese and U.S. scientists.

(4) Seminar on “Fundamentals in Cancer Prevention”
This seminar was held on March 9-10, 1992, at the Maui Marriott Hotel, Lahaina, Maui, Hawaii. The seminar was organized by Drs. Richard Adamson and Peter Greenwald of the National Cancer Institute, Bethesda, Maryland, and Dr. Suketami Tominaga, Aichi Cancer Center Research Institute, Nagoya, Japan. There were seven participants and one observer from Japan and five participants and one observer from the United States.
The purpose of the seminar was to discuss and exchange information on fundamentals in cancer prevention, which has become an important cancer control measure in recent years. Human cancer is thought to develop through multiple stages with involvement of multiple exogenous and endogenous factors. This concept implies the possibility of strategy of primary cancer prevention based on the results of epidemiological studies and experimental studies concerning the etiological factors of human cancer. Epidemiological studies have revealed a close association of causation of human cancer with various environmental factors, especially dietary factors and tobacco smoking. Experimental studies have revealed various mutagens and carcinogens in the environment, especially heterocyclic aromatic amines (HAAs) in cooked meat and fish. Dr. Takashi Sugimura (National Cancer Center, Tokyo), in his introductory remarks, mentioned that (1) cancer prevention was selected as the main theme of the seminar series for the first time, as it is an important emerging field; (2) multiple primary cancer has increased as cured cancer cases have increased because of multiple steps of carcinogenesis, and (3) several new chemicals are now available which could be applied to human cancer prevention-epigallocatechin gallate, new retinoids (AM80, AM580, RE80), Sunphenon, CV3611, decosahexaenoic acid, chorei-tou, etc.).
Dr. Yuzo Hayashi (Biological Safety Research Center, National Institute of Hygienic Sciences, Tokyo) presented the results of three modulating factors in carcinogenesis of lung cancer and colon cancer in animals: (1) promoting effects of cigarette smoking on tracheobronchial tumors in hamsters, (2) enhancement of mouse lung tumors by high fat intake and its retardation by involuntary exercise, and (3) possible participation of prostaglandins in the development of colon cancer in rats. The results of these animal experiments could be applicable to primary cancer prevention in humans.
Dr. Aya Hanai (Department of Field Research, Center for Adult Diseases, Osaka) reviewed recent trends in the cancer incidence and mortality in Japan. The number of cancer cases has increased markedly in recent years mainly reflecting an increase of the aged population, whereas the age standardized cancer incidence rate has increased slightly in males and is almost stable in females. In the same period, the age-standardized mortality of cancer has remained almost unchanged in males and has decreased slightly in females. The discrepancy between the incidence rate and mortality rate was especially large for stomach cancer and uterine cancer which suggested the effects of cancer screening and improvement of diagnostic and therapeutic techniques for cancer. The five year survival rates for major cancers were also reviewed. No major improvement was observed for cancers of the lung, liver, gallbladder, pancreas and esophagus. Future estimation of cancer incidence in Japan predicted relative increases of cancers with poor prognosis and aged cancer cases.
Dr. Shcila Zahm (National Cancer Institute, Bethesda, Maryland) emphasized the importance of occupational studies in cancer prevention since occupational exposures are usually greater, occur more frequently and over longer periods than do general population exposures. Occupational cancers can be prevented by eliminating or reducing workers’ exposures to carcinogenic substances. Although estimates vary, occupational exposure may account for about 5% of all cancers in men and about 2% of all cancers in women in the U.S., totalling 38,350 preventable cancer cases annually.
Dr. Kazuo Iwai (Research Institute of Tuberculosis, Kiyose, Tokyo) presented the results of a risk assessment for human lung cancer due to diesel exhaust particles (DPE) in three different methods. The estimated lifetime lung cancer risk due to DPE was (1) 82.7 x ^-4 calculated from the amount of deposited carbonous dusts in the lung of inhabitants in Tokyo and from the risk of lung cancer according to the deposited DPE in rats, (2) 12.3(5.2-29.5) x 10 ^-4 calculated from the rate of lung burden to the lifetime inhalation dose in humans and rats, assuming the ratio of 3.5 of the rate in the two species, and (3) 4.16(1.56-7.69) x 10^-4 calculated from the results of epidemiological studies on workers exposed to DPE in the occupational environment. Thus, the lifetime lung cancer risks due to DPE estimated from the three different methods were not much different From these results it was further estimated that about 5% of lung cancer in males and about 20% of lung cancer in females were attributable to DPE in urban areas in Japan.
Dr. Larry Kolonel (Cancer Research Center of Hawaii, University of Hawaii at Manoa, Hawaii) reviewed the results of epidemiological studies on dietary factors and cancer prevention. Dietary factors positively related to cancer were fat, total caloric intake, alcohol, aflatoxin, N-nitroso compounds, etc., and factors inversely related to cancer were fruits and vegetables. The responsible components in these foods have been suggested to be beta-carotene and other carotenoids, alpha-tocopherol, vitamins C and E, fiber, certain trace elements, and other non-nutritive constituents. Apart from the control of cigarette smoking, dietary change holds the greatest potential for widespread prevention of cancer. Although solid evidence is not yet available on the effect of dietary change on cancer prevention, many scientists and health officials feel that sufficient knowledge has accumulated to justify the promulgation of recommendations to the public.
Dr. Richard H. Adamson (National Cancer Institute, Bethesda, Maryland) reviewed studies on heterocyclic aromatic amines (HMS). These HAAs occur during the normal process of cooking and, therefore, a large population is exposed to these compounds which are not only mutagenic but also carcinogenic in experimental animals. Methods are available that individuals can take to lessen their intake of HAAs including (1) vary the method of cooking meat poultry and fish; (2) eat beef medium instead of well done; (3) microwave fish and poultry more often; (4) when barbecuing, microwave first and discard the juice in the microwave dish; (5) stew, poach and boil meat and/or fish more often, and (6) do not allow drippings from meat to become dry before making gravy.
Dr. Peter Greenwald (National Cancer Institute, Bethesda, Maryland) reviewed chemoprevention trials in cancer prevention. The National Cancer Institute (NCD) has supported a number of chemoprevention trials and studies on diet and cancer. Chemoprevention studies consist of four steps: (1) basic research to screen chemopreventive agents by in vitro testing; (2) preclinical studies by in vivo animal screening and toxicity testing; (3) clinical trials-some trials are appropriate for a medical setting while others are suitable for the public health setting or unique populations; and (4) application of results obtained from clinical trials to medical practice and the public at large. Chemoprevention is a promising area of research, and one which demonstrates the need for a strong focus on human prevention research at leading cancer centers and universities.
Dr. Akira Oshima (Department of Field Research, Osaka Cancer Prevention and Detection Center, Osaka) reviewed smoking control activities in Japan. In spite of the high prevalence of smoking in male adults in Japan (61.2% in 1992), the national and local governments have not invested much effort or monies on smoking control. Dr. Oshima and his staff have conducted a number of smoking control programs with limited resources: “Osaka Quit Contest,” smoking cessation class, development of the health risk appraisal system for motivating toward smoking cessation, development of a smoking cessation kit for physicians, study for application of the “Know Your Body Program” to the Japanese children, etc. It is hoped these activities will be disseminated widely in Japan and contribute to promotion of smoking control activities in various settings.
Dr. Peter Greenwald reviewed smoking control activities in the United States, where tobacco use is declining but still is responsible for nearly 35%, of all annual cancer deaths. The NCI has supported a number of smoking and tobacco control programs to accelerate the downward trend in national smoking prevalence rates: basic and applied research, clinical and community-based intervention trials, and large-scale demonstration projects. Currently two large smoking control programs are in progress. One is the NCI’s Community Intervention Trial for Smoking Cessation (COMMIT), involving more than two million people in 22 communities. Another is the American Stop Smoking Intervention Study (ASSIST), which was jointly implemented by NCI and the American Cancer Society.
Dr. Margaret Tucker (National Cancer Institute, Bethesda, Maryland) reviewed risk factors and prevention activities for melanoma and skin cancers in the United States. The incidence rates of melanoma and basal cell and squamous cell carcinomas of the skin are increasing in whites in the United States. Individuals at highest risk of developing melanoma are those with a genetic predisposition such as xeroderma pignentosum or dysplastic nevi/melanoma trait. Individuals at highest risk of developing non-melanoma skin cancers are again individuals with a genetic predisposition such as nevoid basal cell carcinoma syndrome or xeroderma pignentosum. However, these genetic predispositions account for a small proportion of all skin cancers. The major environmental risk factor for both melanoma and basal and squamous carcinomas of the skin is sun exposure, although the type of sun exposure differs. Melanoma risk is related to acute intermittent exposure resulting in sunburn, while basal and squamous cell carcinomas are related to total dose of sun over a lifetime. A second major environmental risk factor for both melanoma and non-melanoma skin cancers is immunosuppression, although it accounts for only a small proportion of skins cancers. Thus, the major areas for melanoma and non-melanoma skin cancer prevention is changing the sun exposure habits of the white population.
Dr. Shigeru Hisamichi (Department of Public Health, Tohoku University School of Medicine, Sendai) reviewed cancer screening programs in Japan, where the national and local governments have invested a lot of effort and monies in this effort. Because of the high mortality rates of stomach and cervical cancers in the past the national government has supported stomach and cervical cancer screening programs since around 1965. Under the Health and Medical Service Law for the Aged, which was enacted in 1983, the national government started to support breast and lung cancer screening programs in addition to stomach and cervical cancer screening programs, and is scheduled to support a colorectal cancer screening program beginning in April 1992. These nationwide cancer screening programs have been implemented without fully testing the effectiveness of cancer screening programs. In recent years, epidemiologists have been evaluating the efficacy of cancer screening programs by several epidemiological and statistical methods.
Dr. Suketami Tominaga (Aichi Cancer Center Research Institute, Nagoya) presented the results of an estimate of the potential of future cancer prevention in Japan. The main results were as follows: (1) about 4-8% of cancers could be prevented by smoking control if the smoking prevalence is reduced from 60% (males) and 15% (females) to 50-30% in males and 10-5% in females, (2) about 6-12% of cancers could be prevented by improvement of dietary habits-reduction of salty foods and more intake of raw vegetables, green/yellow vegetables and fiber-rich foods, (3) about 3-6% of cancers could be prevented by control of HBV/HCV infection, (4) another 0-2% of cancers could be prevented by improvement of the work environment and air pollution. A total of 13-28% of cancer incidence/mortality could be prevented if the above mentioned primary prevention activities are promoted extensively; (5) about 10-13% of cancer deaths could be prevented if the participation rate of cancer screening is elevated from about 10% at present to 30% for all cancer screening programs for the stomach, cervix, colorectum, breast and lung, and (6) about 6-10% of cancer mortality could be avoided by application of the state-of-the-art treatment. From the present study it was estimated that a total of 30-50% of cancer deaths/incidence could be avoided if all cancer control activities are promoted extensively in Japan by the year 2000.
Dr. Peter Greenwald concluded the seminar by pointing out several priority areas in cancer prevention research/activities in the future: public guidance, especially toward a smoke-free society, and dietary guidance, cancer prevention trials in a medical setting and in the public health setting, biomakers, biotechnology changes in the food supply, sensitivity to environmental factors, and build cancer prevention into the mainstream of major research institutions. This seminar was the first seminar on prevention in the U.S.-Japan Seminar series. Because of the success of the seminar and importance of cancerprevention in cancer control measures, all participants strongly encouraged another seminar in cancer prevention in a few years to review the progress of cancer prevention research and to promote cancer prevention activities.



SEMINAR AGENDA AND PARTICIPANTS

(1) INVOLVEMENT OF GROWTH REGULATORY GENES IN CANCER
February 15-17, 1992 Maui, Hawaii

AGENDA