Masao Seto, M.D.,D.M.Sc., Chief
Yoshitaka Hosokawa, M.D., D.M.Sc., Section Chief(as of April,1997)
Ryoji Ishida, Ph.D., Senior Researcher(as of April,1996)
Tatsuroh Joh, M.D., D.M.Sc., Researcher
Ritsuro Suzuki, M.D.(as of January,1997)
Keiko Nishida, B.P., Senior Research Assistant
Hiroko Suzuki, B.P., Research Assistant
Miyabi Sugiyama, V.M.D., Research Assistant(until April,1997)
Yumiko Maeda, B.S., Research Assistant(as of April,1997)
Visiting Trainees
Yoshitoyo Kagami, M.D., Department of Hematology and Chemotherapy, Aichi Cancer
Center Hospital
Hirofumi Taji, M.D., Department of Hematology and Chemotherapy, Aichi Cancer
Center Hospital
Jun Takizawa, M.D., The First Department of Internal Medicine, Niigata
University School of Medicine
Masaaki Kume, M.D., The Third Department of Medicine, Akita University School
of Medicine
Tomoaki Akagi, M.D., The First Department of Internal Medicine, Hirosaki
University School of Medicine
Shinsuke Harada, M.D., The Second Department of Internal Medicine, Nagoya City
University School of Medicine
Rika Takashima, B.P., Gifu Pharmaceutical University
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General Summary
Research in this laboratory is aimed at generating a better understanding of the genetic and molecular bases of human cancer, with eventual application of the acquired knowledge in the field of medical oncology. Our work has been mainly focused on hematologic malignancies in cooperation with researchers of the Department of Pathology and Clinical Laboratories (Dr. S. Nakamura), and the Department of Hematology and Chemotherapy (Chief, Y. Morishima) of the Aichi Cancer Center Hospital. Hematologic malignancies have several advantages for studying the molecular bases of neoplasia. Chromosomal abnormalities have been analyzed by a large number of researchers and the observed strong association between specific chromosome changes and specific hematopoietic tumors provides direct evidence that the resultant gene alterations play a pivotal role in the disease development . Over the last two years we have concentrated attention on the 11q13 translocation (associated with mantle cell lymphomas), and the 11q23 translocation (associated with acute myelogeneous leukemias and acute lymphoblastic leukemias of infants). The second advantage of studying hematopoietic malignancies is that the various leukemias and lymphomas have been classified in detail with respect to cell surface markers, developmental lineages and stages, so that they can be used for studying important factors and signals for cell differentiation and proliferation. This line of work includes hunting for new genes which are differentially expressed among various hematopoietic malignancies. Indeed, any gene which is differentially expressed could play a role in hematopoietic cell differentiation and proliferation, and therefore carcinogenesis, and in this respect, the hematopoietic malignancies offer very useful tools to explore new insights.
The development of reagents for both research and clinical use is a high priority and we have produced monoclonal antibodies against BCL-1/cyclin D1 products, one of which, 5D4, has proven to be very specific for diagnosis of mantle cell lymphoma, a very malignant disease. The application of this antibody allowed definition of a new molecular pathologic entity, a subset of MCL with a poor prognosis, that should be treated with a new strategy including bone marrow transplantation.
Through analysis of clinical samples accumulated in Aichi Cancer Center Hospital, we have identified a distinct new hematolymphoid disease entity designated as myeloid/natural killer cell precursor acute leukemia.
Another line of inquiry is focussed on DNA topoisomerase II as a molecular target of antitumor drugs. For improved cancer chemotherapy, we have investigated whether the M phase checkpoint is involved in susceptibility of cells to the ICRF-193, a catalytic inhibitor of topoisomerase II. Intriguingly, Serine-1212 of topo II was found to be phosphorylated only in the M phase, and the enzyme was concentrated in centromeres in metaphase.
1. A Small Deletion in the 3'-Untranslated Region of the
Cyclin D1/PRAD1/ BCL-1 Oncogene in a Patient with Chronic Lymphocytic Leukemia
Hosokawa, Y., Suzuki, R., Joh, T., Maeda, Y. Nakamura, S.*1, Kodera,
Y.*2, Arnold, A.*3 and Seto, M.
The cyclin D1/PRAD1 oncogene, a
key regulator of the G1 phase of the cell cycle, has been widely incriminated
in the pathogenesis of human neoplasia. Cyclin D1 was also demonstrated to be
identical to the long sought BCL-1 oncogene in B cell malignancies with the
t(11;14)(q13;q32) translocation. The cyclin D1 gene can be deregulated by
different mechanisms, including chromosome inversion, translocation, and gene
amplification. We report here a small deletion in the 3'-untranslated portion
of the cyclin D1 gene in leukemia cells of a patient diagnosed with B-CLL,
associated with overexpression of the corresponding cyclin D1 mRNA. During a
Northern blot survey of B cell malignancies, we identified a patient whose CLL
cells showed a marked increase in a 1.5-1.6kb cyclin D1 mRNA species; this
transcript appeared to be somewhat smaller than the commonly observed 1.7 kb
mRNA, reminiscent of findings for a human breast cancer cell line, MDA MB-453
in which the 3'-untranslated region of the cyclin D1 gene is truncated,
resulting in an increased stability of the cyclin D1 mRNA. Subsequent Southern
blot analysis showed the genomic DNA from the patient's cells to contain an
extra band in the EcoRI digest, suggesting alteration in one allele of the
cyclin D1. Polymerase chain reaction (PCR) analysis of the genomic DNA and
direct DNA sequencing clearly disclosed that one allele of the cyclin D1 gene
is deleted in the 3'-untranslated region, which would contribute to an increased
stability of its mRNA. Reverse transcription-polymerase chain reaction (RT-PCR)
analysis and direct DNA sequencing revealed that the cyclin D1 mRNA is deleted
in the corresponding region. Although the deleted regions of the cyclin D1 gene
in the CLL patient and the MDA MB-453 cell line contain no typical AUUUA mRNA
instability sequence, they were found to encompass AU-rich sequences. Indeed,
it was demonstrated that in the MDA MB-453 cell line, the truncated mRNA is
more stable than the normal transcript after actinomycin D treatment. Thus, the
AU-rich sequence in the deleted region appears to contribute to the rapid
degradation of the cyclin D1 mRNAs. The present study provides the first
example of a clonal deletion within the transcribed noncoding region of the
cyclin D1 gene in a primary patient sample, indicating that the deletion took
place in the primary malignancy and not during the process of cell line
establishment. This finding provides further evidence of a critical role for
cyclin D1 in the pathogenesis of B cell malignancies and highlights a novel
mechanism, a small deletion in the 3'-untranslated region, responsible for
deregulation of the cyclin D1 gene in oncogenesis.
*1 Department of Pathology and Clinical Laboratories, Aichi Cancer
Center Hospital
*2 Department of Internal Medicine, Japanese Red Cross Nagoya First
Hospital
*3 Endocrine Unit, Massachusetts General Hospital
2. Clinicopathologic Study of PRAD1/cyclin D1
Overexpressing Lymphoma with Special Reference to the Mantle Cell Lymphoma, A
Distinct Molecular Pathologic Entity
Yatabe, Y*1, Nakamura, S.*1, Seto, M., Kuroda, H.,
Kagami, Y.*2, Suzuki, R., Ogura, M.*2, Kojima, M., *3,
Koshikawa, T.*1, Ryuzo Ueda, R. *4 and Taizan Suchi, T.*1.
Mantle cell lymphomas (MCLs) are
frequently associated with the overexpression of PRAD1/cyclin D1 activated by
11q13 translocation and its molecular consequence with BCL-1 gene
rearrangement. We recently described positive nuclear staining monoclonal
antibody against a PRAD1/cyclin D1 product to be correlated with mRNA
overexpression in MCLs. In this study, we have immunohistochemically
investigated the PRAD1/cyclin D1 protein in a large series of 334
lymphoproliferative disorders, including 39 cases of MCLs. Based on the cyclin
D1 positivity, CD5 expression and morphologic features of the tumor tissue,
four groups of MCL-related lesions were identified among the B-cell lymphomas
examined: 36 cases with cyclin D1 overexpression, 35 (95%) of which exhibited
CD5-positivity and MCL-morphology (Group I); 4 cases of lymphomas with
MCL-morphology and CD5 expression, but lacking cyclin D1 overexpression (Group
II); 4 cases of lymphomas without cyclin D1 overexpression and surface CD5, but
falling within the morphologic boundaries of MCLs (Group III); 11 cases of
CD5-positive diffuse large cell lymphomas without cyclin D1 overexpression
(Group IV). The cases of Group I demonstrated quite homogeneous
clinicopathologic features, identical to those of MCLs. This group showed a
poor prognosis (11% 5 year survival), in pronounced contrast with that of Group
II (100%). Although the four groups of MCL-related lesions sometimes
demonstrated overlapp in their histologic and/or phenotypic spectra, each
appeared to show distinct clinicopathologic and prognostic profiles. Our study
provides a basis for further clarification of the natures of the neoplasms of
Groups II, III and IV. Moreover, this comprehensive studyprovided an indication
that overexpression of PRAD1/cyclin D1 is biologically essential to
define MCLs.
*1 Department of Pathology and Clinical Laboratories, Aichi Cancer
Center Hospital
*2 Department of Hematology and Chemotherapy, Aichi Cancer Center
Hospital
*3 Department of Pathology and Clinical Laboratories, Ashikaga Red
Cross Hospital
*4 The Second Department of Internal Medicine, Nagoya City
University School of Medicine
3. Somatic Hypermutations in the VH Segment
of Immunoglobulin Genes of CD5 Positive Diffuse Large B-Cell Lymphomas
Kume, K., Suzuki, R., Yatabe, Y.*1, Kagami, Y.*2, Suzuki,
H., Miura, I.*3, Miura, A.B.*3, Morishima, Y.*2,
Nakamura, S.*1and Seto, M.
The de novo CD5-positive (CD5+)
diffuse large B-cell lymphoma (DLBL) has been recently identified as
constituting a homogeneous subgroup in terms of clinicopathologic and genotypic
characteristics, but its developmental stage origin remains to be elucidated.
Previous sequence analysis of the variable region of the immunoglobulin heavy
chain (VH) showed that cells of CD5+ B-cell malignancies,
such as the mantle cell lymphoma(MCL) and the B-cell chronic lymphocytic
leukemia (B-CLL), represent pre-germinal center (GC) stage B cells in contrast
to the post-GC stage of most DLBLs, which show somatic hypermutations in VH
genes. In the present study, we investigated the VH sequence of de
novo CD5+ DLBL to clarify whether this entity is pre-GC stage, as
other CD5+ B-cell malignancies, or post-GC stage, as typical of
DLBL. All eight cases (four CD5+ DLBL and four CD5-negative (CD5-)
DLBL) examined showed somatic hypermutations in the VH segment and
two of the CD5- DLBL cases showed intra-clonal diversity, suggesting
derivation from the same maturation stage as CD5- DLBL, distinct
from the other indolent CD5+ B-cell lymphomas, B-CLL and MCL. These
data suggest that de novo CD5+ DLBLs do not merely lie within a
continuous spectrum, together with B-CLL and MCL, but rather that they
represent a biologically distinct variant within the diagnostic framework of
diffuse large B-cell lymphoma.
*1 Department of Pathology and Clinical Laboratories, Aichi Cancer
Center Hospital
*2 Department of Hematology and Chemotherapy, Aichi Cancer Center
Hospital
*3 The Third Department of Internal Medicine, Akita University
School of Medicine
4. Molecular Cytogenetic Delineation of the Breakpoint
at 18q21.1 in a NHL Cell Line (Karpas 1106) Derived from a Mediastinal B-cell
Lymphoma
Akagi, T., Tamura, A.*1, Nakamura, S.*2, Karpas, A.*3,
Silverman, G.A.*4, Morishima, Y.*5, Taniwaki, M.*1
and Seto, M.
The breakpoint of the 18q21
translocation of the B-cell non-Hodgkin's lymphoma (NHL) cell line Karpas 1106P
was delineated by fluorescence in situ hybridization (FISH). Karpas 1106P,
derived from a mediastinal B-cell lymphoma, shows an immunophenotype
characteristic of the marginal zone B-cell lymphoma (MZL): smIg+,
pan-B antigen+, CD5-, CD10-, and CD23-. The original G-banded
karyotype displayed a complex translocation containing
t(X;13;18)(q28;q12.1;q21). Double-color FISH (DCFISH) with whole chromosome
painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast
artificial chromosome (YAC) clones defined t(X;13;18) as
ider(X)t(X;18;13)(q28;q12q21.1;q12.1). The immunoglobulin heavy chain (IgH)
gene was not involved in the chromosomal translocation as detected by DCFISH
with VH and Cg gene probes. By using contiguous YAC clones mapped from 18q12.3 to
q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1. In
Karpas 1106P, the distal part of chromosome 18 from the breakpoint
(18q21.1-qter) was found to be deleted, showing loss of heterozygosity of this
region. In addition, the chromosomal segment 18q21.1 was duplicated and
inserted to ider(X)t(X;13;18) between Xq28 and 13q12.1, with maintenance of its
original orientation. The DNA sequence of the breakpoint region contained in
y852H2 can be expected to serve as a candidate locus for further molecular
dissection to identify the causative gene of MZL.
*1 Third Department of Internal Medicine, Kyoto Prefectural
University of Medicine
*2 Department of Pathology and Clinical Laboratories, Aichi Cancer
Center Hospital
*3 Department of Haematology, University of Cambridge, MRC Centre
*4 Department of Pediatrics, Harvard Medical School
*5 Department of Hematology and Chemotherapy, Aichi Cancer Center
Hospital
5. Chimeric MLL Products with a Ras Binding Cytoplasmic
Protein AF6 Involved in t(6;11)(q27;q23) Leukemia Localize in the Nucleus
Joh, T., Kagami, Y.*1, Kakuda, H.*2, Sato, T.*2,
Yamamoto, T.*3, Takahashi, To.*4, Ueda, R.*5,
Kaibuchi, K.*3 and Seto, M.
Analysis of 11q23 translocations, commonly observed in infantile leukemias and therapy-related leukemias, has revealed the presence of the MLL/ALL-1/HRX gene encoding a 432 kDa protein with AT-hook DNA-binding motifs at 11q23. The MLL gene also has zinc finger motifs which together have been named the PHD finger or the LAP domain. The zinc finger motifs and the C-terminal portion of MLL show significant homology to Drosophila trithorax gene. A cysteine-rich region showing high homology to the mammalian DNA methyltransferase, located on the N-terminal side of the zinc finger motifs has also been noted. However, the biological role of MLL in mammalian cells and the mechanism of leukemogenesis remain to be elucidated. In leukemias with 11q23 translocations, MLL is disrupted and the N-terminal portion, including the AT-hook motifs and the DNA methyltransferase homology region, is fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL. Although more than ten different chromosomal translocation partner genes have been identified, no shared common structure has been recognized among the partner gene products. We previously demonstrated that the chimeric MLL products MLL-LTG9 and MLL-LTG19, as well as MLL-Zf(-), the N-terminal portion common to various chimeric MLL products, localize in nuclei. These data suggest that the N-terminal portion of the MLL gene caused by truncation might play a pivotal role in nuclei leading to leukemogenesis, although a possible contribution of the partner gene region cannot be disregarded.
AF6, one fusion partner of the MLL gene, has recently been
identified as a GTP-Ha-Ras binding protein. It has a Ras-interacting domain and
a Dlg homology repeat (DHR) motif which is thought to function in the formation
of protein complexes at the junction of the plasma membrane and the
cytoskeleton. Since the chimeric MLL-AF6 gene found in the
t(6;11)(q27;q23) translocation includes all of AF6 except its N-terminal 35
amino acids, leaving both the Ras-interacting domain and DHR motif intact, identification
of where the MLL-AF6 chimeric products localize in the cell is important for
elucidation of the mechanism of leukemogenesis resulting from MLL
translocations. Therefore, we examined the subcellular localization of MLL-AF6
transiently expressed in COS7 cells and in a CTS cell line with a
t(6;11)(q27;q23) translocation derived from an acute myeloid leukemia patient.
Immunofluorescence staining data and cell fractionation analyses demonstrated
MLL-AF6 chimeric products to localize in the nuclei despite the fact that AF6
itself localizes in the cytoplasm, confirming the importance of the nuclear
localization of chimeric MLL products. Furthermore, in order to clarify the
region of the N-terminal portion of MLL responsible for nuclear localization, we
generated deletion mutants and AT-hook motifs were found to be most important.
*1 Department of Hematology and Chemotherapy, Aichi Cancer Center
Hospital
*2 Department of Pediatrics, School of Medicine, Chiba University
*3 Division of Signal Transduction, Nara Institute of Science and
Technology
*4 Laboratory of Immunology
*5 The Second Department of Internal Medicine, Nagoya City
University School of Medicine
6. CD7+ and CD56+ Myeloid/Natural
Killer Cell Precursor Acute Leukemia: A Distinct Hematolymphoid Disease Entity
Suzuki, R., Seto, M., Morishima, Y.*1 and Nakamura, S.*2
The French-American-British Cooperative Group (FAB) classification has been employed as a standardized criteria for acute leukemia based on morphological characteristics, but it does not specify the cell lineage of the leukemic cells, and immunological classifications have also been introduced. We have identified a previously unrecognized type of acute leukemia, of conceivable myeloid and NK cell precursor phenotype.
The leukemic cells of this type of leukemia coexpress NK cell associated CD56 antigen, myeloid associated CD33 antigen, and stem cell associated CD7 and CD34 antigens. No other T- or B-cell specific antigens have been found to be positive except in one CD2 positive case. Three of seven cases weakly expressed cytoplasmic CD3, although none expressed surface CD3 antigen. Clinically, striking extramedullary involvement was evident at initial presentation with peripheral lymphadenopathy and/or mediastinal masses. Two cases lacked any leukemic cells in the bone marrow at diagnosis, and one case presented with a relatively low percentage of leukemic cells in bone marrow for a diagnosis of acute leukemia. Less than 3% of the leukemic cells demonstrated positive cytochemical myeloperoxidase staining. Morphologically, the cells were generally L2-shaped, with variation in cell size, round to moderately irregular nuclei with prominent nucleoli, pale cytoplasm, and a lack of azurophilic granules. Histopathological examination of biopsied specimens of extramedullary tumors revealed a lymphoblast-like morphology, implying a problem in differential diagnosis from lymphoblastic lymphomas, especially in cases lacking bone marrow involvement.
By Southern blot analysis, all but one presented germline configurations of the T-cell receptor b and g chain genes and immunoglobulin heavy chain gene. By Northern blot analysis, CD3 and CD3e chain gene mRNA was detected in two out of four cases and six out of six cases, respectively.
The therapeutic response of the cases showed their myeloid characteristics. Three patients were successfully treated with chemotherapy for acute myeloid leukemia (AML), whereas three other patients proved refractory to chemotherapy for lymphoid malignancies, although two responded to subsequent AML chemotherapy. However, in spite of intensive chemotherapy including allogeneic bone marrow transplantation, most cases relapsed and pursued fatal courses.
In conclusion, the described
cases of myeloid/NK cell precursor acute leukemia feature common cytologic,
genetic and clinicopathologic characteristics of immature myeloid and NK cell
lineage, and might constitute a distinct biological and clinical disease
entity. Such a close relationship between myeloid and T/NK cell lineage is
contrary to the concept of common lymphoid progenitors between T-cell and
B-cell lineages and might suggest that the genealogy table of the hematopoietic
system requires revision.
*1 Department of Hematology and Chemotherapy, Aichi Cancer Center
Hospital
*2 Department of Pathology and Clinical Laboratories, Aichi Cancer
Center Hospital
7. DNA Topoisomerase II as a Molecular Target of
Antitumor Drugs: Mitotic Checkpoint Generated under Inhibition of Topoisomerase
II-mediated Chromosome Segregation and Modulation of Biological Activity of the
Enzyme by Phosphorylation
Ishida, R., Nishida, K., Hamatake, M.*1, Takashima, R.*2,
Haraguchi, T.*3, Hiraoka, Y.*3, Yano, T.*4
,Shibata, M.*4, Iguchi, K.*2, Hirano, K.*2 and
Seto, M.
Cells have checkpoints during the cell cycle to ensure that events do not occur until the previous process has been completed. This is important for maintaining the integrity of the genetic material and the relationship between checkpoints and susceptibility of cells to antitumor drugs has therefore attracted a great deal of attention. ICRF-193, a catalytic topoisomerase II (topo II) inhibitor, delays the progression from metaphase to anaphase, but not from anaphase to G1 phase, suggesting that its effects are related to an inability for chromosome segregation through inactivation of topoII activity. The ICRF-193-induced delay is presumably related to a checkpoint because the drug does not delay the progression in mitotic CHO cells , which are checkpoint deficient. Since ICRF-193 kills mitotic cells most effectively, it is an intriguing possibility that the M phase checkpoint may be involved in their susceptibility to the drug. To confirm that this is the case, we isolated M-phase checkpoint proficient and deficient Balb-3T3 cells, and compared the susceptibility of these cells to ICRF-193. When cells were exposed to colcemid for 48 h, the proficient cells accumulated 4C DNA while the deficient cells had both 4 C and 8 C DNA. To determine whether the proficient and deficient cells exhibit distinct responses to ICRF-193, the effects of the drug on cell cycle progression in the M phase was examined in the two types of cells. Cell cycle progression from metaphase to G1 phase was inhibited by ICRF-193 in the proficient cells, but not in deficient cells. The proficient cells were also more sensitive to the cytotoxic effects of ICRF-193 than the defient cells when they were exposed to the drug during mitosis or under asynchronized conditions. The different susceptibility to the cytotoxicity was not due to variation in topo II activity since this and chromosome segregation of proficient and deficient cells were inhibited by the same concentration of ICRF-193. Moreover, the inability to segregate chromosome itself did not lead to be lethal. We can conclude that differences in events following the inhibition of chromosome segregation, possible M-phase checkpoints, are responsible for the distinct responses of the two cells to ICRF-193.
To investigate the relation between the modulation of activity and subcellular localization of topo II in cell cycle, we have prepared three monoclonal antibodies which recognize immunogenic phosphoepitopes but not other phosphoepitopes or non-phosphorylated forms of the peptide. The phosphorylated sites are located at serine-29, serine-1212 and threonine-1342 of human topo II and the antibodies reacted with purified topo II and anti-topo II immunoprecipitants. Western blot analysis revealed anti PT 1342 antibody recognition of topo II in all cell cycle phases, while anti PS 1212 antibody only demonstrated binding during mitosis. Immunostaining of phosphorylated topo II confirmed that the former antibody stained interphase nuclei, but the latter did not. The phosphorylated topo II was located in the nuclear scaffold in the pro- and prometaphases, centromeres in metaphase and then the nuclear scaffold in ana- and telophases. Since topo II begins to locate to centrosomes in pro- and prometaphases, it is likely that mitotic phosphorylation of serine-1212 of topo II is involved in this change of localization to centromeres.
Since many
antitumor drugs kill tumors through induction of apoptosis, their effects are
presumably enhanced if they are used in combination with other drugs which
impinge on apoptotic processes. To clarify the biological events involved in
induction of apoptosis, we have examined associated changes in proteins by
antitumor drugs. When Molt-4 cells were exposed to etoposide, ICRF-193 and
neocarzinostatin, they exhibited apoptotic cell death determined by flow
cytometry using FITC-labeled annexin V staining of phosphatidylserine on
membranes by and detection of hypo-diploid cells. Following induction of
apoptosis, a low molecular weight protein identified as thymosin b4 by HPLC
analysis, was commonly decreased. Thus its down-regulation may be involved
antitumor drug-induced apoptosis.
*1 Department of Pharmacology
*2 Gifu Pharmaceutical University
*3 Kansai Advanced Research Centre
*4 Medical and Biological Laboratories