Molecular studies of acute myeloid leukemia and the telomerase reverse transcriptase gene
Author: Lindvall, Charlotta
Date: 2002-09-20
Location: Hudklinikens föreläsningssal, Karolinska Sjukhuset
Time: 9.00
Department: Institutionen för molekylär medicin / Department of Molecular Medicine
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Thesis (814.5Kb)
Abstract
Multiple chromosome rearrangements (MCRs) are detected in approximately 10% of patients with acute myeloid leukemia (AML), and are associated with an adverse prognosis. Comprehensive analysis of the chromosome rearrangements in these complex karyotypes has previously been hampered by the limitations of conventional cytogenetic techniques such as G-banding. As a consequence, our knowledge concerning the genetic alterations in these malignancies is limited. We applied spectral karyotyping (SKY), comparative genomic hybridization (CGH) and cDNA microarrays to bone marrow cells from AML patients with MCRs in order to characterize these rearrangements on the genomic and transcriptional level.
Using SKY and CGH we resolved 12 complex AML karyotypes, and also detected novel chromosome rearrangements. We showed that many of the deletions scored by G-banding harboured cryptic chromosome rearrangements. Ile majority were unbalanced translocations and most frequently resulted in chromosome loss of 5q, 7q and 17p, and chromosome gain of 11q. In addition, the SKY analysis revealed a number of balanced translocations that had not been described before. Some seem recurrent and may reflect novel fusion genes directly involved in leukemogenesis. (Paper I)
The chimeric transcripts of one reciprocal translocation detected by SKY, a t(8;16)(p11;p13);(MOZCBP), was analyzed at the sequence level using RT-PCR. The resulting RT-PCR method could be diagnostically useful, since the detection of t(8;16) in AML has clinical ramifications and may be difficult to identify by chromosome banding alone. (Paper II)
In order to investigate the transcriptional profiles of leukemic cells with MCRs we also developed and applied a high-density cDNA microarray assay. We showed that most of the chromosome rearrangements were manifested in aberrant gene expression profiles in a gene-dosage dependent manner. (Paper III)
We also investigated the role of hTERT/telomerase in tumorigenesis and disease. Telomeres, which constitute the ends of chromosomes and are essential for genomic stability and integrity, are synthesized by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase consists of an RNA template, a reverse transcriptase (hTERT) and telomerase associated proteins. Because hTERT/telomerase activation is critical for cellular immortalization and tumorigenesis we investigated the copy numbers of hTERT in human tumors and found that hTERT is a frequent target for DNA amplification. (Paper IV)
Chri-du-Chat syndrome (CdCs) is one of the most common human deletion syndromes and results from a deletion of the distal part of chromosome arm 5p, where the hTERT gene is located. We showed that a heterozygous loss of hTERT is present in CdCs and that hTERT is limiting for telomere maintenance in humans. Therefore, CdCs might be used as a model to study hTERT regulation and telomerase biology in humans. (Paper V)
Certain somatic cells can acquire an immortal phenotype by forced expression of hTERT/telomerase. Treatment with exogenous hTERT has been proposed as a cell-based therapy to allow indefinite expansion of normal human cells without damaging their genomes. However, using cDNA microarrays we showed that the gene expression profile of hTERT-immortalized fibroblasts (hTERT-BJ cells) is significantly different from that of normal mortal fibroblasts. One of the highly expressed genes in the hTERT-BJ cells encodes epiregulin and we found its expression was required to maintain the immortal phenotype. Given the significant difference in gene expression profiles between the normal and hTERT-immortalized fibroblasts and the dose relationship between epiregulin and tumorigenesis, we concluded that the use of hTERT for expansion of normal human cells for therapeutic purposes must be approached with great caution. (Paper VI)
Using SKY and CGH we resolved 12 complex AML karyotypes, and also detected novel chromosome rearrangements. We showed that many of the deletions scored by G-banding harboured cryptic chromosome rearrangements. Ile majority were unbalanced translocations and most frequently resulted in chromosome loss of 5q, 7q and 17p, and chromosome gain of 11q. In addition, the SKY analysis revealed a number of balanced translocations that had not been described before. Some seem recurrent and may reflect novel fusion genes directly involved in leukemogenesis. (Paper I)
The chimeric transcripts of one reciprocal translocation detected by SKY, a t(8;16)(p11;p13);(MOZCBP), was analyzed at the sequence level using RT-PCR. The resulting RT-PCR method could be diagnostically useful, since the detection of t(8;16) in AML has clinical ramifications and may be difficult to identify by chromosome banding alone. (Paper II)
In order to investigate the transcriptional profiles of leukemic cells with MCRs we also developed and applied a high-density cDNA microarray assay. We showed that most of the chromosome rearrangements were manifested in aberrant gene expression profiles in a gene-dosage dependent manner. (Paper III)
We also investigated the role of hTERT/telomerase in tumorigenesis and disease. Telomeres, which constitute the ends of chromosomes and are essential for genomic stability and integrity, are synthesized by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase consists of an RNA template, a reverse transcriptase (hTERT) and telomerase associated proteins. Because hTERT/telomerase activation is critical for cellular immortalization and tumorigenesis we investigated the copy numbers of hTERT in human tumors and found that hTERT is a frequent target for DNA amplification. (Paper IV)
Chri-du-Chat syndrome (CdCs) is one of the most common human deletion syndromes and results from a deletion of the distal part of chromosome arm 5p, where the hTERT gene is located. We showed that a heterozygous loss of hTERT is present in CdCs and that hTERT is limiting for telomere maintenance in humans. Therefore, CdCs might be used as a model to study hTERT regulation and telomerase biology in humans. (Paper V)
Certain somatic cells can acquire an immortal phenotype by forced expression of hTERT/telomerase. Treatment with exogenous hTERT has been proposed as a cell-based therapy to allow indefinite expansion of normal human cells without damaging their genomes. However, using cDNA microarrays we showed that the gene expression profile of hTERT-immortalized fibroblasts (hTERT-BJ cells) is significantly different from that of normal mortal fibroblasts. One of the highly expressed genes in the hTERT-BJ cells encodes epiregulin and we found its expression was required to maintain the immortal phenotype. Given the significant difference in gene expression profiles between the normal and hTERT-immortalized fibroblasts and the dose relationship between epiregulin and tumorigenesis, we concluded that the use of hTERT for expansion of normal human cells for therapeutic purposes must be approached with great caution. (Paper VI)
List of papers:
I. Lindvall C, Nordenskjold M, Porwit A, Bjorkholm M, Blennow E (2001). Molecular cytogenetic characterization of acute myeloid leukemia and myelodysplastic syndromes with multiple chromosome rearrangements. Haematologica. 86(11): 1158-64.
Pubmed
II. Panagopoulos I, Isaksson M, Lindvall C, Bjorkholm M, Ahlgren T, Fioretos T, Heim S, Mitelman F, Johansson B (2000). RT-PCR analysis of the MOZ-CBP and CBP-MOZ chimeric transcripts in acute myeloid leukemias with t(8;16)(p11;p13). Genes Chromosomes Cancer. 28(4): 415-24.
Pubmed
III. Lindvall C, Furge K, Bjorkholm M, Guo X, Blennow E, Nordenskjold M, Tean Teh B (2002). Combined genetic- and transcriptional profiling of acute myeloid leukemia with complex and normal karyotype. [Manuscript]
IV. Zhang A, Zheng C, Lindvall C, Hou M, Ekedahl J, Lewensohn R, Yan Z, Yang X, Henriksson M, Blennow E, Nordenskjold M, Zetterberg A, Bjorkholm M, Gruber A, Xu D (2000). Frequent amplification of the telomerase reverse transcriptase gene in human tumors. Cancer Res. 60(22): 6230-5.
Pubmed
V. Zhang A, Zheng C, Hou M, Lindvall C, Erlandsson F, Bjorkholm M, Gruber A, Blennow E, Xu D (2002). Delection of the telomerase reverse transcriptase gene and haploinsufficiency of telomerase in Cri-du-Chat syndrome. [Submitted]
VI. Lindvall C, Hou M, Tomurasaki T, Zheng C, Henriksson M, Bjorkholm M, Gruber A, Tean Teh B, Nordenskjold M, Xu D (2002). Molecular characterization of hTERT-immortalized human fibroblasts by gene expression profiling: activation of the epiregulin gene. [Manuscript]
I. Lindvall C, Nordenskjold M, Porwit A, Bjorkholm M, Blennow E (2001). Molecular cytogenetic characterization of acute myeloid leukemia and myelodysplastic syndromes with multiple chromosome rearrangements. Haematologica. 86(11): 1158-64.
Pubmed
II. Panagopoulos I, Isaksson M, Lindvall C, Bjorkholm M, Ahlgren T, Fioretos T, Heim S, Mitelman F, Johansson B (2000). RT-PCR analysis of the MOZ-CBP and CBP-MOZ chimeric transcripts in acute myeloid leukemias with t(8;16)(p11;p13). Genes Chromosomes Cancer. 28(4): 415-24.
Pubmed
III. Lindvall C, Furge K, Bjorkholm M, Guo X, Blennow E, Nordenskjold M, Tean Teh B (2002). Combined genetic- and transcriptional profiling of acute myeloid leukemia with complex and normal karyotype. [Manuscript]
IV. Zhang A, Zheng C, Lindvall C, Hou M, Ekedahl J, Lewensohn R, Yan Z, Yang X, Henriksson M, Blennow E, Nordenskjold M, Zetterberg A, Bjorkholm M, Gruber A, Xu D (2000). Frequent amplification of the telomerase reverse transcriptase gene in human tumors. Cancer Res. 60(22): 6230-5.
Pubmed
V. Zhang A, Zheng C, Hou M, Lindvall C, Erlandsson F, Bjorkholm M, Gruber A, Blennow E, Xu D (2002). Delection of the telomerase reverse transcriptase gene and haploinsufficiency of telomerase in Cri-du-Chat syndrome. [Submitted]
VI. Lindvall C, Hou M, Tomurasaki T, Zheng C, Henriksson M, Bjorkholm M, Gruber A, Tean Teh B, Nordenskjold M, Xu D (2002). Molecular characterization of hTERT-immortalized human fibroblasts by gene expression profiling: activation of the epiregulin gene. [Manuscript]
Issue date: 2002-08-30
Rights:
Publication year: 2002
ISBN: 91-7349-291-4
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