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1.
Int J Hematol ; 77(5): 463-70, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12841384

ABSTRACT

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) -gammadelta T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCRgammadelta T-cells were CD8alphabeta, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.


Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Promoter Regions, Genetic , T-Lymphocytes/cytology , Thymus Gland/cytology , WT1 Proteins/physiology , Animals , Antigens, CD/analysis , CD8 Antigens/analysis , CD8-Positive T-Lymphocytes/cytology , Cell Count , Cell Differentiation , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, gamma-delta , T-Lymphocyte Subsets/cytology , WT1 Proteins/genetics
2.
Cancer Sci ; 94(3): 271-6, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12824921

ABSTRACT

The expression levels of the Wilms' tumor gene WT1 were examined in 36 cases of various types of human bone and soft-tissue sarcomas using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). They included 12 malignant fibrous histiocytomas (MFH), 3 malignant peripheral nerve sheath tumors (MPNST), 6 synovial sarcomas (SyS), 4 myxoid liposarcomas (MyLS), one angiosarcoma (AGS), one clear cell sarcoma (CCS), and 9 osteosarcomas (OS). Eleven (92%) of 12 MFH, 2 (67%) of 3 MPNST, all (100%) of 6 SyS, 2 (50%) of 4 MyLS, one AGS, one CCS, and 5 (56%) of 9 OS cases overexpressed WT1 in the range of 1.4 x 10(-3)-3.9 x 10(-1) levels (WT1 expression level in K562 leukemic cells was defined as 1.0). Thus, 28 (78%) out of 36 various types of human bone and soft-tissue sarcomas overexpressed the WT1 gene. Immunohistochemical analysis showed positive staining for WT1 protein in all of 4 cases (one case each of MFH, MyLS, AGS and OS) with WT1 gene overexpression detected by RT-PCR analysis, demonstrating clearly that WT1 was expressed at the protein level in various types of human bone and soft-tissue sarcomas. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of 10 exons of the WT1 gene in 8 different sarcoma samples (3 MFH, one SyS, one MyLS, one AGS, and 2 OS). The present study demonstrates that various types of human bone and soft-tissue sarcomas frequently overexpress the wild-type WT1 gene, suggesting an important role of the wild-type WT1 gene in tumorigenesis of various types of human bone and soft-tissue sarcomas.


Subject(s)
Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Wilms Tumor , Sarcoma/genetics , WT1 Proteins/genetics , Adult , Aged , Base Sequence , Bone Neoplasms/classification , Bone Neoplasms/pathology , DNA Primers , Female , Humans , Immunohistochemistry , Kidney Neoplasms/genetics , Male , Middle Aged , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma/classification , Sarcoma/pathology , WT1 Proteins/analysis , Wilms Tumor/genetics
3.
Cancer Immunol Immunother ; 51(11-12): 614-20, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12439606

ABSTRACT

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.


Subject(s)
HLA-A Antigens/metabolism , Neoplasms/therapy , Peptide Fragments/immunology , T-Lymphocytes, Cytotoxic/immunology , WT1 Proteins/immunology , Humans , Immunotherapy , Tumor Cells, Cultured , WT1 Proteins/genetics , WT1 Proteins/metabolism
4.
Int J Cancer ; 100(3): 297-303, 2002 Jul 20.
Article in English | MEDLINE | ID: mdl-12115544

ABSTRACT

Expression of the Wilms' tumor gene WT1 in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WT1 gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WT1 protein with an anti-WT1 antibody. Overexpression of the WT1 gene was also demonstrated in 5/6 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WT1 gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the nonmutated, wild-type WT1 gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WT1 gene and its products.


Subject(s)
Lung Neoplasms/genetics , WT1 Proteins/genetics , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Small Cell/genetics , Carcinoma, Small Cell/pathology , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Mutation , Neoplasm Staging , RNA Splicing
5.
Br J Haematol ; 116(2): 409-20, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11841446

ABSTRACT

The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT-PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1.3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT-PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1.1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1.2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+CD38- and CD34+CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38- and lineage-committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells.


Subject(s)
Antigens, CD34 , Hematopoietic Stem Cells/metabolism , Leukemia/genetics , WT1 Proteins/genetics , Flow Cytometry/methods , Gene Expression , Humans , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction
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