Age is the largest single risk factor for the development of cancer in mammals. Age-associated chromosomal changes, such as aneuploidy and telomere erosion, may be vitally involved in the initial steps of tumorigenesis. However, changes in gene expression specific for increased aneuploidy with age have not yet been characterized. Here, we address these questions by using a panel of fibroblast cell lines and lymphocyte cultures from young and old age groups. Oligonucleotide microarrays were used to characterize the expression of 14,500 genes. We measured telomere length and analyzed chromosome copy number changes and structural rearrangements by multicolor interphase fluorescence in situ hybridization and 7-fluorochrome multiplex fluorescence in situ hybridization, and we tried to show a relationship between gene expression patterns and chromosomal changes. These analyses revealed a number of genes involved in both the cell cycle and proliferation that are differently expressed in aged cells. More importantly, our data show an association between age-related aneuploidy and the gene expression level of genes involved in centromere and kinetochore function and in the microtubule and spindle assembly apparatus. To verify that some of these genes may also be involved in tumorigenesis, we compared the expression of these genes in chromosomally stable microsatellite instability and chromosomally unstable chromosomal instability colorectal tumor cell lines. Three genes (Notch2, H2AFY2, and CDC5L) showed similar expression differences between microsatellite instability and chromosomal instability cell lines as observed between the young and old cell cultures suggesting that they may play a role in tumorigenesis.
The discovery of tyrosine kinases that, once deregulated, can cause malignancy, allowed the development of specifically acting anti-cancer compounds. In chronic myeloid leukaemia (CML), the Bcr-Abl kinase inhibitor imatinib (STI571, Gleevec) induces impressive response rates. However, resistance occurs especially in advanced phase CML and Ph+ ALL, primarily as a consequence of point mutations within the Bcr-Abl kinase domain that prevent imatinib from binding. To overcome imatinib resistance, alternative Abl kinase inhibitors are finding their way into clinical trials. However, it is likely that resistance to second-generation compounds will occur as well. Therefore, it will be critical to determine specific resistance profiles for each particular compound. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of Bcr-Abl resistance mutations emerging in the presence of imatinib or an alternative Abl-kinase inhibitor. Using this strategy, the findings in inhibitor resistant sublines reflect observations made in CML patients with imatinib resistance, including Bcr-Abl mutations, amplification of the Bcr-Abl gene, and overexpression of the Bcr-Abl protein. We here provide a detailed methodological description, and discuss the implications of this strategy for different clinically relevant oncogenic tyrosine kinases.
The Abl-tyrosine kinase inhibitor Imatinib efficiently targets the Bcr-Abl kinase and produces major cytogenetic responses in most patients with chronic phase CML. In contrast, patients with advanced stage CML or Ph+ ALL frequently become refractory to Imatinib treatment. Resistance arises predominantly from point mutations in the Abl-kinase region, Bcr-Abl amplification or clonal evolution due to secondary genetic aberrations. To screen for genes contributing to clonal evolution, we have employed retroviral insertional mutagenesis in a murine CML/ALL model to identify potential candidate genes leading to Imatinib resistance. We found proviral insertions near the RUNX3/AML2 promoter in Imatinib resistant leukemic clones, leading to upregulation of RUNX3 mRNA expression. To analyze the effects of high RUNX3 levels on Imatinib response, we expressed RUNX3 in a Bcr-Abl-transformed murine pre-B-cell line. Significantly, whereas there was no effect on Imatinib-mediated proliferation inhibition, the cells displayed a marked reduction of apoptosis. A RUNX3R193A mutant carrying a mutation in the DNA-binding domain of RUNX3 did not protect from apoptosis, indicating that transcriptional regulation by RUNX3 was required to induce the anti-apoptotic effects. To allow for a controlled activation of RUNX transcriptional activity and to extend our analysis to other members of the RUNX family of transcription factors, we constructed 4-OH-tamoxifen (TAM) inducible RUNX3/AML2- and RUNX1/AML1-Estrogen receptor (ER) fusion proteins. These fusion proteins readily translocated from the cytoplasm into the nucleus and activated a RUNX-dependent TCRß-luciferase construct upon addition of TAM. Using these constructs, we could demonstrate that activation of RUNX3 as well as RUNX1 protected Bcr-Abl-transformed Ba/F3 cells from Imatinib-induced apoptosis. Furthermore, we found that RUNX1 mRNA levels were significantly upregulated in patients with Ph+ ALL upon resistance development. Taken together, our data indicate that elevated RUNX3 or RUNX1 levels may contribute to Imatinib resistance in Bcr-Abl expressing leukemic cells.
<div>Abstract<p>Age is the largest single risk factor for the development of cancer in mammals. Age-associated chromosomal changes, such as aneuploidy and telomere erosion, may be vitally involved in the initial steps of tumorigenesis. However, changes in gene expression specific for increased aneuploidy with age have not yet been characterized. Here, we address these questions by using a panel of fibroblast cell lines and lymphocyte cultures from young and old age groups. Oligonucleotide microarrays were used to characterize the expression of 14,500 genes. We measured telomere length and analyzed chromosome copy number changes and structural rearrangements by multicolor interphase fluorescence <i>in situ</i> hybridization and 7-fluorochrome multiplex fluorescence <i>in situ</i> hybridization, and we tried to show a relationship between gene expression patterns and chromosomal changes. These analyses revealed a number of genes involved in both the cell cycle and proliferation that are differently expressed in aged cells. More importantly, our data show an association between age-related aneuploidy and the gene expression level of genes involved in centromere and kinetochore function and in the microtubule and spindle assembly apparatus. To verify that some of these genes may also be involved in tumorigenesis, we compared the expression of these genes in chromosomally stable microsatellite instability and chromosomally unstable chromosomal instability colorectal tumor cell lines. Three genes (<i>Notch2, H2AFY2</i>, and <i>CDC5L</i>) showed similar expression differences between microsatellite instability and chromosomal instability cell lines as observed between the young and old cell cultures suggesting that they may play a role in tumorigenesis.</p></div>
<div>Abstract<p>Age is the largest single risk factor for the development of cancer in mammals. Age-associated chromosomal changes, such as aneuploidy and telomere erosion, may be vitally involved in the initial steps of tumorigenesis. However, changes in gene expression specific for increased aneuploidy with age have not yet been characterized. Here, we address these questions by using a panel of fibroblast cell lines and lymphocyte cultures from young and old age groups. Oligonucleotide microarrays were used to characterize the expression of 14,500 genes. We measured telomere length and analyzed chromosome copy number changes and structural rearrangements by multicolor interphase fluorescence <i>in situ</i> hybridization and 7-fluorochrome multiplex fluorescence <i>in situ</i> hybridization, and we tried to show a relationship between gene expression patterns and chromosomal changes. These analyses revealed a number of genes involved in both the cell cycle and proliferation that are differently expressed in aged cells. More importantly, our data show an association between age-related aneuploidy and the gene expression level of genes involved in centromere and kinetochore function and in the microtubule and spindle assembly apparatus. To verify that some of these genes may also be involved in tumorigenesis, we compared the expression of these genes in chromosomally stable microsatellite instability and chromosomally unstable chromosomal instability colorectal tumor cell lines. Three genes (<i>Notch2, H2AFY2</i>, and <i>CDC5L</i>) showed similar expression differences between microsatellite instability and chromosomal instability cell lines as observed between the young and old cell cultures suggesting that they may play a role in tumorigenesis.</p></div>
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