(1) Preneoplastic Lesions / Precancerous Lesions
Kauai, Hawaii (February 13-15,1998)
The purpose of this meeting was to explore the emerging concepts of preneoplasia in the context of modern molecular biology and genetics. In many ways, preneoplasia is the next great frontier in molecular genetics as we try to explore the fundamental changes that initiate the inevitable progress to human malignancy. While a great deal of research has gone into unraveling the myriad of genetic events present in invasive tumors and cell lines, only recently have we begun to look for these genetic changes in preneoplastic lesions. Molecular genetic studies of preneoplastic lesions are hampered by the difficulty in identifying these lesions either morphologically or clinically. Moreover, there is a necessity to carefully isolate often small microscopic lesions followed by fine microdissection for analysis of the involved epithelial cells. These limitations have been overcome by significant improvements in clinical detection, pathologic microdissection techniques, and molecular biology. We thus gathered an interested group of scientists from the United States and Japan to explore emerging areas of particular interest in preneoplasia.
Dr. Setsuo Hirohashi explored genetic and epigenetic alterations in the development of human hepatocell carcinoma. Dr. Hirohashi identified key genetic events in precursor lesions identified pathologically. He was able to provide a rational order of genetic events in the development of human hepatocarcinogenesis. Dr. Fiichi Tahara followed with a similar development of progression model in the development of various types of stomach cancer. In addition to tumor suppressor gene alterations, it appears that amplifications and proto-oncogene activation are also significant components in the genetic progression of stomach cancer. Moreover, different genetic changes may predict the ultimate morphology in stomach cancer. Dr. Jerry Shay proceeded with an overview of telomerase in preneoplasia and recent data suggesting that the reintroduction of the reverse transcriptase component of telomerase led to cellular mortalization. Abundant discussions ensued about the importance of telomerase in immortalization and its critical role in cancer. It became apparent that many, if not most, tumors displayed telomerase activation in the preneoplastic state.
Dr. Adi Gazdar presented a spectrum of genetic changes in the progression of lung cancer. He was particularly intrigued by the large patches of preneoplastic cells in the lung that displayed similar genetic alterations. The concept of allele-specific loss and their clonality was discussed in detail. Dr. Yoichi Konishi presented genetic studies in preneoplastic lesions of the pancreatic duct in hamsters. This model seem to reflect the genetic changes present in primary tumors and provided a general order of genetic events for pancreatic cancer progression. Dr. William Grizzle then summarized known genetic and epigenetic in prostate cancer. He addressed issues concerning the true preneoplastic component of prostate cancer including adenomatous hyperplasia and prostatic intraepithelial neoplasia (PIN).
Dr. David Sidransky showed evidence to support the clonal origin of multiple tumors in the head and neck area and evidence for the presence of large preneoplastic patches in these patients. He then reiterated recent studies demonstrating that microsatellite changes could be used for the clonal detection of human cancers in various bodily fluids. Dr. Ryuzo Ueda followed with substantial data on the evaluation of minimal residual disease in hematological malignancies. He received, various RT- PCR approaches for the detection of minimal residual cells and their clinical implications. Dr. Brian Reid described genetic alterations in the progression of esophageal cancer. He also outlined the occurrence of early genetic changes years before the progression of cancer in patients with Barrett’s esophagus and the clonal expansion of large patches of cells before the development of invasive cancer.
Dr. Takao Sekiya demonstrated new approaches for the detection of genetic and epigenetic abnormalities in cancer cells. These types of approaches appear to be promising and add to the list of possible new approaches for the isolation of deleted and methylated areas in cancer genomes. Dr. Darryl Shibata related work describing the progression of genetic alterations in colorectal cancer. He further elucidated the use of microsatellite instability as a molecular clock and the role of this approach in determining the relative progression of various preneoplastic lesions. Dr. Ramon Parsons described the cloning of the PTEN gene located on chromosome 10q in his laboratory. Moreover, he addressed the frequency of PTEN alterations in various tumor types including gliomas and prostate cancer.
Dr. Kenji Shimiizu described genetic alterations of the pIO7-E2F4 cell cycle regulation in human tumors. Specifically, he concentrated on alterations of mismatch repair genes by microsatellite instability as well as potential activation of E2F4 in human cancers. Dr. Doug Brash elucidated the known models of skin cancer progression and the role of p53 in this pathway. He described the early emergence of p53 mutations followed by clonal expansion in the progression of skin cancer. Finally, Dr. Akira Horii discussed the importance of “gatekeeper” and “caretaker” genes in the progression of preneoplasia. He described his work on mismatched repair genes including recent work on gastrointestinal cancers. He identified critical targets of microsatellite instability including a group of genes whose coding regions are a frequent targets of mutation for the mismatch repair system. These changes lead to the progression of various gastrointestinal cancers.
In summary this was an excellent and probing meeting that begins to outline the genetic progression of preneoplasia in various types of human cancer. There was strong emphasis on molecular work supporting the concepts of genetic progression and clonal evolution in the development of human cancer. It was agreed by all that further efforts need to concentrate on further elucidation of these genetic changes, trying to understanding which are critical for the initiation of clonal evolution and which are essential for the initial progression of various preneoplastic lesions. It is clear from this meeting that elucidation of the genetic changes will provide now targets for early detection and elucidation of these changes may also lead to novel prognostic assays. Eventually, preventive or chemopreventive approaches may help to decrease the burden of the preneoplastic lesions and eventually the overall burden of cancer in our society. There was strong enthusiasm by all participants for continued basic science and clinical development of the important fundamental concepts discussed at this meeting.
I would like to thank you for allowing us to organize this meeting. It was a pleasure to work with Dr. Hirohashi and I look forward to working with him in future U.S.-Japan cooperative scientific endeavors.
PARTICIPANTS
UNITED STATES
Ramon Parsons, M.D., Ph.D.
Department of Pathology
Columbia University
Room 14-453 630 W.
168th Street, New York, NY 10032
Adi F. Gazdar, M.D.
Hamon Cancer Center, University of Texas
Southwestern Medical Center
5323 Harry Hines Boulevard
Dallas, TX 75235-8593
Stanley R. Hamilton, M.D.
Department of Pathology
Johns Hopkins University School of Medicine
632 Ross Research Building
720 Rutland Avenue, Baltimore, MD 21205-2196
Brian J. Reid, Ph.D., M.D.
Public Health Science Division
Fred Hutchinson Cancer Research Center
1124 Columbia Street, CI-015
Seattle, WA 98104
Darryl K. Shibata, M.D.
Department of Pathology
LAC-USC Medical Center
Unit #1 , Room 2428, 1200 N. State Street
Los Angeles, CA 90033
Stephen N. Thibodeau, Ph.D.
Molecular Genetics Laboratory
Mayo Clinic
Rochester, MN 55905
William E. Grizzle,, Ph.D., M.D.
Department of Pathology
University of Alabama
University Station Birmingham, AL 35294-0007
David Sidransky, M.D.
Division of Head and Neck Cancer Research
Department of Otolaryngology-Head & Neck Surgery
818 Ross Research Building
720 Rutland Avenue, Baltimore, MD 21205-2196
JAPAN
Dr. Setsuo Hirohashi
Deputy Director
National Cancer Center Research Institute
5-1-1 , Tsukiji, Chuo-ku, Tokyo 104-0045
Dr. Eiichi Tahara
Professor
Hiroshima University School of Medicine
1-2-3, Kasumi, Minami-ku, Hiroshima 734-0037
Dr. Yoichi Konishi
Professor
Cancer Center, Nara Medical University
840, Shijo-cho, Kashihara-shi, Nara 634-0813
Dr. Takao Sekiya
Chief
National Cancer Center Research Institute
5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045
Dr. Ryuzo Ueda
Professor
Nagoya City University Medical School
1, Aza-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-0001
Dr. Kenji Shimizu
Professor
Institute of Cellular and Molecular Genetics
Okayama University Medical School
2-5-1 , Shikata-cho, Okayarna 700-0914
Dr. Akira Horii
Professor
Tohoku University School of Medicine
2-1 , Seiryo-machi, Aoba-ku, Sendai-shi, Miyagi 980-0872
Observer:
Dr. Masayuki Noguchi
Professor
Institute of Basic Medical Sciences, University of Tsukuba
1-1-1, Tennoudai, Tsukuba-shi, Ibaraki 305-0006
(2) Angiogenesis and Cancer
(NO Report Received)
Organized by Dr. Napoleone Ferrare and Masabumi Shibuya
PARTICIPANTS
JAPAN
Dr. Kaoru Miyazaki
Department of Cell Biology, Institute for Biological Research
Yokohama City University
641-12, Maioka-cho, Totsuka-ku
Yokohama, Knagawa 244-0913,
Dr. Kohei Miyazono
Department of Biochemistry
The Cancer institute, Japanese Foundation for Cancer Research
1-37-1, Kami-ikebukuro Toshimaku
Tokyo, 170-9455
Dr. Shin-ichi Nishikawa
Department of Molecular Genetics, Faculty of Medicine, Kyoto University
Shogoin, Kawahara-cho, 53
Sakyo-ku, Kyoto, 606-8397
Dr. Tetsuo Noda
Department of Cell Biology,
The Cancer institute, Japanese Foundation for Cancer Research
1-37-1, Kami-ikebukuro, Toshima-ku
Tokyo, 170-8455
(Tohoku Univ. Sch. of Med., Dept. of Molecular Genetics)
Dr. Motaharu Seiki
Department of Cancer Cell Research
The Institute of Medical Science, The University of Tokyo
4-6-1, Shirokanrdai, Minato-ku, Tokyo, 108-8639
Dr. Masaburni Shibuya
Department of Genetics
The Institute of Medical Science, The University of Tokyo
4-6-1 , Shirokanedai, Minato-ku, Tokyo, 108-8639
Dr. Masaaki Terada
National Cancer Center Research Institute
5-1-1, Tsukiji, Chuo-ku, Tokyo, 104-0045
Dr. Shoichiro Tsukita
Department of Cell Biology, Kyoto University, Faculty of Medicine
Yosbida-konoe, Sakyo-ku, Kyoto, 606-8501
UNITED STATES
Dr. Noel P. Bouck
Department of Microbiology
Immunology and R. H. Luric Cancer Center
Northwestern University Medical School
Chicago, IL, 60611
Dr. David A. Cheresh
Departments of Immunology and Vascular Biology
The Scripps Reseach Institute
La Jolla, CA 92037
Dr. Vishva M. Dixit
Department of Molecular Oncology
Genentech, Inc., I DNA Way
South San Francisco, CA 94080
Dr. Napoleone Ferrara
Department of Cardiovascular Research.
Genentech, Inc.
South San Francisco, CA 94080
Dr. Rekesh K. Jain
Department of Radiation Oncology
Harvard Medical School, Massachusetts General Hospital
Boston, MA 02114
Dr. Robert S. Keirbel
Cancer Biology Research, Division of Biological Sciences
Sunnybrook Health Science Center, and Department of Medical Biophysics
University of Toronto, Toronto, Ontario, CANADA
Dr. Jan E. Schnitzer
Department of Pathology, Harvard Medical- School
Beth Israel Deaconess Medical Center
Boston, MA 02215
Dr. Richard I. Weiner
Department of Obstetrics, Gynecology and Reproductive Sciences
University of California School of Medicine
San Francisco, CA 94143
(3) Cell Cycle Control and Cancer
A meeting on Cell Cycle Control and Cancer was convened on February 20-21, 1998 at Maui, Hawaii, under the auspices of the US/Japan Cooperative Cancer Research Program and organized by Snorri Thorgeirsson (National Cancer Institute, USA) and Hiroto Okayama (University of Tokyo, Japan). Cancer is best described as a mass of cells with highly elevated, uncontrolled proliferative potential, which is caused by mutations in cellular growth control genes that are partly inherited and partly generated by spontaneous as well as environmental DNA damage. Consequently, understanding the system controlling cell proliferation is essential for unveiling the molecular mechanism of carcinogenesis. In this joint seminar, experts studying cell cycle start control and related mechanisms in both countries presented their latest research outcomes for extensive discussion and exchanges of information. The aim of this joint seminar was to help deepen our understanding of the highly complex mechanism controlling cell proliferation to better characterize the fundamental mechanism underlying malignant transformation. In addition, this meeting provided an excellent opportunity for the participants to engage in mutual assistance and planning future collaborations. Robert Weinberg (Massachusetts Institute of Technology, USA) presented the opening lecture on cell immortalization and tumorigenesis. The fact that cancer cells show replicative immortality in vitro and express telomerase enzyme has suggested the possibility that telomerase expression is causally important for the acquisition of this most important cell phenotype. To address this question directly, Weinberg and his colleagues isolated the EST2 gene that specifies the catalytic subunit of the telomerase in yeast. The sequence of EST2 and that of the Euplotes gene reported by the Czech Laboratory were then used to isolate the human homolog, which was referred to as hEST2. Expression of this gene is upregulated when mammalian cells become immortal. This observation is compatible with the notion that the expression of this gene suffices for the acquisition of enzyme activity in these cells. Indeed, Weinberg showed that when hEST2 was ectopically expressed in mortal human cells they exhibited catalytic activity.
RECESSIVE ONCOGENES
Mutational inactivation of both alleles of the RB (retinoblastoma protein) gene is the rate-limiting step in retinoblastoma formation, but mutations of BR are frequently seen in other tumor types during tumor progression. RB has been shown to have pleiotropic functions, including cell cycle regulation, cell differentiation, apoptosis, and genomic stability. Using a RB knockout model, Eva Y.-H. Lee (Institute of Biotechnology, University of Texas Health Science Center, USA) described studies demonstrating how RB inactivation affects different cell types. RB-/- mouse embryos die during days 12- 16 of gestation with abnormalities in multiple cell types including erythrocytes. Lee used transplantation of fetal hematopoietic cells to assess the developmental defects of RB-/-cells. While there is increased proliferation of cells of erythrocyte lineage, most blood cell lineages matured normally and RB-1- recipients lived for more than a year without evidence of erythroleukemia. Therefore, RB mutation may not be a rate-limiting step in malignant transformation of leukemia. This is in accordance with the limited tumor types developed in the human carriers and mouse RB+/- heterozygotes.
Le also described identification of a novel neuron enriched , LxCxE-containing protein that interacts with RB, and a novel neuron-specific gene, RB-inhibited gen (Rig-1), whose expression is elevated in the RB-deficient embryos. Expression of Rig-1 promoter in activated in neuronal cells and is repressed by RB protein. Lee also showed that the transmembrane form of Rig-1 is capable of repressing the neuron-specific Tal promoter and increased S-phase progression in cultured cells. Lee hypothesized that deregulated Rig-1 activity may generate at least some of the neuron phenotypes seen in the BR knockout mice.
One effective approach to identify novel oncogenes and recessive oncogenes is to take advantage of a relatively large collection of Drosophila mutants with properties of hyperplasia or neoplasmic overgrowth and isolate their homologs. Hideyuki Saya (Kumamoto University School of Medicine, Japan) has been taking this approach and described the isolation and properties of human homologs of the discs large (dig), neurolized and warts tumor suppressor genes. The human dig, termed NE-dig, is located on chromosome Xq13 and encodes a 100 kDa protein that is predominantly expressed in non-proliferating cells in various tissues. Overexpression of NE-dlg suppresses proliferation of various cancer cells and concomitantly induces apoptosis in them. NE-dig protein interacts with the C-terminal of APC tumor suppressor protein and with the newly identified protein called nedasin. Human neurolized gene was mapped between D10S192 and DIOS566 on chromosome 10q25.1, a region frequently deleted in astrocytomas. In addition, expression of this gene is highly diminished or totally lost in many glioblastomas and astrocytomas. Moreover, the U251MG glioma cell line has a point mutation in neurolized gene. Since in Drosophila loss-of-function mutations of this gene cause hyperplasia of primitive neuronal cells, these findings suggest that human neurolized is a good candidate for a brain-specific tumor suppressor gene.
CELL CYCLE START AND ARREST CONTROL
RB and p53 are phospho-proteins regulating cell proliferation. It has been well established that the function of RB, a negative regulator of the cell cycle start, itself is negatively regulated by phosphorylation that is catalyzed by various Cdk/cyclin complexes. Yoichi Taya (National Cancer Center Research Institute, Tokyo, Japan) described that various Cdks (cyclin-dependent kinases) phosphorylate RB, but on different sites. Ser780 is phosphorylated by Cdk4-cyclin D, but not by Cdk2-cyclin E, and conversely, Thr356 is phosphorylated by Cdk2-cyclin E, but not by Cdk4-cyclin D. Interestingly, among all potential phosphorylation sites in RB, Thr821 is most efficiently phosphorylated by Cdc2-cyclin B. The biological significance of this differential phosphorylation is currently unclear, but it might modulate the target specificity of RB protein. In contrast to RB, the physiological role of phosphorylation of p53 has not been established. Taya presented data showing that p53 undergoes phosphorylation at Ser15 after DNA damage, which leads to reduced interaction with its negative regulator, MDM2. One good candidate for the kinase phosphorylating Ser15 is DNA-Pk, since it can phosphorylate this site in vitro. In addition, he found that Cdk-activating kinase, which is required for cell cycling, transcription and DNA repair, phosphorylates Ser33 of p53. Thus, p53, DNA repair and transcription may be coordinately regulated via these kinases.
Cell cycle arrest by the cytokine TGF-!
!!requires inhibition of GI Cdks. This can result from the up-regulation of the Cdk inhibitor p15Ink4B, which specifically targets cyclin D-dependent kinases Cdk4 and Cdk6 and allows increased binding of p27 to Cdk2. Antonio Iavarone (Memorial Sloan Kettering Cancer Center, USA) reported that this is not the only mechanism available to TGF-!!!for the inhibition of GI Cdks. Iavarone showed that in the absence of p15Ink4B, TGF-!!!can still inhibit Cdk4 and Cdk6 by increasing their level of tyrosine phosphorylation. Indeed, tyrosine phosphorylation and inactivation of Cdk4 and Cdk6 result from the ability of TGF-!!!to repress the expression of the Cdk-activating phosphatase Cdc25A. Inhibition of Cdc25A expression by TGF-!!!was shown to be at the transcriptional level and a fragment of 589 bp (from -460 to +129 relative to the Cdc25A transcription start site) was sufficient to mediate TGF-!!!induced inhibition. Sequence analysis of the Cdc25A promoter identified “ubiquitous” transcription factor binding sites (one AP2, two SPI, two inverted CCAAT boxes) and two E2F sites, one located 60 bp upstream of the transcription start site (E2F-1) followed by a consensus CHR (cell cycle homology region) and the other spanning the transcription start site (E2F-11). Iavarone further reported that mutational analysis of specific transcription factor binding sites showed: (i) the two SPI sites, the CHR site and the E2F-11 site played no significant role in the efficiency of basal transcription or in the repression by TGF-!!!; (ii) mutation of the E2F-I site did not affect the basal activity, but completely abolished the inhibition of the Cdc25A promoter by TGF-!!!. Furthermore, ectopic expression of E2F/IDP1 relieved repression of the wild-type Cdc25A promoter by TGF-!!!, but expression of E2F4/DPI was only minimally effective. Therefore, the data presented by Iavarone indicate that down-regulation of Cdc25A mRNA expression results from the active repression of Cdc25 promoter by TGF-!!!. Inactivation of the growth inhibitory activity of TGF-!
!!1 is an important step in the neoplastic development of numerous tumor types. Snorri S. Thorgeirsson (National Cancer Institute, USA) described a novel gene, BOG, recently identified and shown to be over-expressed in several transformed rat liver epithelial (RLE) cell lines resistant to the growth-inhibitory effect of TGF-!!!1, as well as in primary liver tumors. Sequence homology searches demonstrated that BOG shares some homology with HPV16E7 and contains the RB binding motif LxCxE. Thorgeirsson demonstrated by using the yeast two-hybrid system and co-immunoprecipitation that BOG binds to RB and that in vivo BOG/RB complexes do not contain E2F1. Furthermore, BOG can displace E2F1 from E2F1/RB complexes in vitro. Overexpression of BOG in non-transformed TGF-!!!1-sensitive RLE cell lines conferred resistance to the growth-inhibitory effect of TGF-!!!1 and rapidly transformed RLE cells that form hepatoblastoma-like tumors when transplanted into nude mice. Thorgeirsson hypothesized that BOG may be important in the transformation process, due in part, to its capacity to confer resistance to the growth-inhibitory effects of TGF-!!!1 through interaction with RB and the subsequent displacement of E2F1. One critical phenotype that distinguishes malignant from benign cells is the ability to start the cell cycle despite the absence of anchorage. Hiroto Okayama (University of Tokyo. Japan) has been taking a unique approach to understanding the mechanism underlying the anchorage-independent cell cycle start. Based on the analysis of recessive NFLK rat fibroblast mutants refractory to transformation by EGF+TGF-!
!!, he concluded that oncogenic signals are qualitatively different from the regular mitogenic signals and that oncogenic signals are able to induce the cell cycle start despite the absence of anchorage. During the investigation of its molecular mechanism, he found that Cdk6-cyclin D is specifically involved in this unique process. When GI-arrested NRK cells were stimulated with serum, their entry into S phase was blocked by microinjection of anti-Cdk4 antibody. In sharp contrast, when the same cells were stimulated with serum containing EGF+TGF-P, their S phase entry was blocked only by microinjection of both anti-Cdk4 and anti-Cdk6 antibodies. All the mutants refractory to transformation by EGF+TGF-!!!behaved like cells stimulated by the regular mitogenic signals, indicating that oneogenic signals facilitate the utilization of Cdk6 by cells for the cell cycle start. In vitro kinase assays, however, suggested that despite such a distinction, both Cdk4 and Cdk6 were activated irrespective of whether cells were stimulated by mitogenic or oncogenic signals. These results suggest that a factor specifically activated by Cdk6 might be present in the cells and that oncogenic signals might make this hypothetical factor available for the cells to use for entry into S phase. These findings may provide a molecular basis for oncogenic signal-induced anchorage-independent cell cycle start. RB and its relatives, p107 and p130, are known to suppress growth of mammalian cells. Growth suppression is thought to occur through the repression of key transcription factors at specific times during the cell cycle. One target of these factors is the E2F transcription factor, which is thought to play a role in progression into the S phase of the cell cycle. Brian Dynlacht (Harvard University, USA) described recent studies on the mechanisms through which RB and p107 prevent S phase entry. In these studies a cell-free, in vitro-reconstituted transcription assay was used to analyze how RB negatively regulates E2F trans-activation. The results suggest a distinct role and protein requirements for RB in blocking a rate- limiting step(s) for E2F activation. Of particular interest was the data showing that the RB-related protein, p107, although able to repress E2F-mediated activation in vitro, can also potently block cell cycle progression by a different mechanism altogether, namely, through the inhibition of associated cyclin-dependent kinases, cyclin AICdk2 and cyclin E/Cdks. Dynlacht suggested that parallels could be drawn between p107 function and the function of certain cyclin-dependent kinase inhibitors (CKls).
One possible mechanism of tumor suppression by TGF-!
!!may involve direct action of TGF-!!!on carcinoma cells. It has recently been reported that TGF-!!!promotes a spindle cell morphology and more aggressive behavior in carcinoma cells that have lost the growth-inhibitory response. That TGF-!!!’s can induce actin stress fiber formation altering cell shape and motility has been known for many years. Retention of the ability to undergo cell shape changes in response to TGF-!!!in cells likely to have mutations that disrupt the growth-inhibitory response suggests the existence of more than one signaling pathway from the TGF-!!!receptors. The pathway for alterations in cell shape and motility probably involves Rho proteins. Harold Moses (Vanderbilt Cancer Center, Vanderbilt University School of Medicine, USA) described studies showing that the cytoplasmic domain of the type I TGF-!!!receptor binds to and phosphorylates farnesyltransferase-, the regulatory subunit for famesyltransferase and type 1 geranyltransferase. Furthermore, Moses showed that the C3 exotransferase of C. botulinum, a specific inhibitor of Rho function, blocks TGF-!!!1 induction of actin stress fibers in MvlLu cells and that TGF-!!!1 increases the abundance and prenylation of RhoB. Moses hypothesized that signaling from the TGF-!
!!receptors involves multiple pathways with some of the signaling molecules mediating growth control being different from those mediating cell shape and motility changes. Genetic data from Drosophila and C. elegans model systems and mutational analysis of human tumors present compelling evidence for the involvement of Smad proteins in TGF-!!!signaling, probably in the growth control pathway. He suggested that his data demonstrating the involvement of Rho proteins in TGF-!!!signaling to modify cell shape and motility processes may be important in relation to invasion and metastasis.ACTIVATION OF REPLICATION ORIGINS
One central question regarding the initiation of the cell cycle is how origins of replication are activated to start DNA synthesis. Recent budding yeast research resulted in the identification of many factors essential for this cellular process. The Orc protein complex, composed of the Orcl-6 subunits, recognizes and binds origins of replication. Cdc6 protein, induced in late GI through early S, together with RLF-B, plays a crucial role in subsequent loading of the Mcm protein complex (Mcm2-7) onto the replication origins. Mcm has been proposed to act as a licensing factor that ensures one firing of each origin in one S phase. DNA polymerases!
!!, !
!!and are then loaded onto the origins and start DNA synthesis. But, how these polymerases, Pol!!! in particular, are loaded remains poorly understood. Haruhiko Takisawa (Osaka University, Japan) described the isolation of a Xenopus hormolog of Cdc45 and presented data that led him to propose that it is a key factor for loading Pol!!!onto chromatin. Budding yeast Cdc45 is essential for the initiation of DNA synthesis and interacts genetically with Mcm5, Mcm7 and Orc. He found that Xenopus Cdc45 protein co-localizes with Pol!!!on chromatin during the initiation of DNA replication. The budding yeast S. cerevisiae constitutes an important model organism for studies on the components and dynamics of DNA replication complexes. In S. cerevisiae, the in vivo chromatin structure of DNA replication origins changes as cells become competent for DNA replication, suggesting that GI phase-specific association of replication factors with origin DNA regulates entry into S phase. Stephen P. Bell (Massachusetts Institute of Technology, USA) described studies in which he used chromatin immunoprecipitation to demonstrate that ORC, Mcm proteins (Mcm4p and Mcm7p), and Cdc45p are components of origin DNA complexes in G1 phase cells. The Mcm protein-origin association is dependent upon ORC and Cdc6p. He also showed that during S phase, Mcm proteins dissociate from the origin complexes and associate with non-origin DNA. DNA Pol!
!!, a replicative DNA polymerase present at replication forks, exhibits a similar pattern of DNA association during S phase, and both Mcm-DNA and Pol a-DNA associations are similarly affected by slowing replication elongation. Based on these data, he suggested that Mcm proteins are components of eukaryotic replication forks. Unwinding of double-helical DNA is prerequisite for polymerases to synthesize DNA. Therefore, one important question regarding origin activation is how replication origins are unwound toward the onset of DNA replication. Yukio Ishimi (Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan) found a clue to answer this question. He described that the Mcm4, 6, 7 complex possesses a DNA helicase activity, whereas Mcm2 functions to load the Mcm4, 6, 7 complex onto chromatin. Mcm2 protein contains a nuclear localization signal and has the ability to bind histone. Both ATPase and DNA helicase activities co-purified with a 600 kDa protein complex containing equal amounts of Mcm4, 6 and 7 whereas Mem2 and other Mcms co-sedimented more slowly than the Mcm4, 6, 7 complex in glycerol gradient centrifugation. Addition of Mcm2 protein to the Mcm4, 6, 7 complex inhibited its helicase activity in a dose-dependent manner, attaining complete inhibition with an equal molar amount. This finding raises the possibility that Mcm2 may also play a regulatory role in the DNA helicase activity of the Mcm4, 6, 7 complex. Despite some structural similarities, Mcm2 and Mcm4, 6, 7 appear to play radically different roles in the initiation of DNA synthesis. Recent studies show that many factors essential for S phase onset are well conserved throughout eukaryotes. In fact, structural and functional homologs for the budding yeast Orc, Mcm and Cdc6 have been isolated from human and other organisms. Budding yeast Cdc7 kinase is another factor essential for S phase onset, though its molecular role is not well understood. Like cyclin-dependent kinases, Cdc7 requires Dbf4, a non-catalytic subunit, for its activity. Dbf4 is induced at the GI/S boundary whereas Cdc7 is constitutively expressed. Hisao Masai (Institute of Medical Science, University of Tokyo, Japan) described isolation of homologs for Cdc7 and Dbf4 from fission yeast and mammals. The Dbf4 homologs are essential for the full activity of the corresponding Cdc7 homolog kinases. Microinjection of anti-human Dbf4 antibody into a primary human fibroblast blocked its entry into S phase. Like other S phase-essential genes, the mouse Dbf4 gene contains two E2F-responsive cis-elements in its promoter. These findings provide further evidence for evolutionary conservation of the system controlling the onset of S phase.
RECOMBINATION AND NUCLEAR TRANSPORT
Recombinational repair is a major cellular repair pathway for double-strand breaks (DSBS) of chromosomal DNA. This pathway is conserved from yeast to mammals. Rad51, being a homologue of E. coli RecA, is a key component of the recombinational repair system. While Rad5l is dispensable for proliferation in lower eukaryotes, attempts to generate murine ES cells with disrupted Rad5l failed, suggesting that Rad5l may be essential for the proliferation of higher eukaryotes. To define the role of Rad5l in higher eukaryotes, Shunichi Takeda (Kyoto University, Japan) generated conditional Rad5l-deficient cells from the chicken cell line DT40 by targeted integration of a repressible promoter-driven human Rad5l gene. He presented data showing that Rad5l is essential for cell proliferation because of the spontaneous generation of DSBS during proliferation. Upon repression of the trans-gene, the Rad5l-deficient cells accumulated in G2 with some cells undergoing mitosis before cell death. Chromosomal analysis revealed that they had isochromatid-type breaks (1.5/cell), which were distributed randomly. This observation indicates that a small number of random DSBs occurred spontaneously during the cell cycle of higher eukaryotes. This illuminates the importance of the DSB repair system for the maintenance of genetic integrity during proliferation of higher eukaryotes. DSB repair defects might underlie chromosome instability associated with tumorigenesis.
One of the cellular processes that have to be considered for the control of the cell cycle is nuclear transport, because the nucleus is the critical place for cell cycle events to take place. Some nuclear proteins contain the nuclear localization signal, whereas some others contain the nuclear export signal (NES). Among well characterized proteins having NES are mitotic cyclin and M4P kinase. Minoru Yoshida (University of Tokyo, Japan) has been studying the molecular target for an anti-tumor antibiotic leptomycin B and recently found that Crml is a key component of the nuclear export machinery whose function is inhibited by leptomycin B. Crml was initially identified from a fission yeast mutant. Leptomycin B binds to Cnnl in vitro, and microinjection of an anti-Crml antibody specifically blocks export of coinjected NES-bearing proteins from nuclei. The second antibiotic Yoshida described is trichostatin A (TSA), an inhibitor of histone deacetylase. Histone deacetylase has recently drawn attention because it is involved in transcriptional control. This enzyme plays a role in RB-mediated transcriptional repression. He found that TSA induces expression of p21Cki independently of p53, suggesting that histone deacetylase is also involved in the control of p21 expression. This in turn suggests that TSA might be useful for cancer therapy. Indeed, recent experiments show that FR901228, an inhibitor of histone deacetylase, has a potent antitumor activity. As described above, the latest experimental results on 1) telomerase in cell immortalization; 2) cell cycle arrest and start control; 3) TGF- pathway; 4) function of RB and newly identified recessive oncogenes; 5) activation of replication origins; 6) recombination and nuclear transport were presented and extensively discussed. This meeting advanced our knowledge, and also illuminated several critical questions to be answered in order to understand the molecular mechanisms underlying oncogenic transformation and cell cycling.
(This meeting report has been published in the Japanese Journal of Cancer Research, 89: 1093-1097, October 1998)