| 1. |
- Gisselsson Nord, David, et al.
(författare)
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Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity
- 2000
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 97:10, s. 5357-5362
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Tidskriftsartikel (refereegranskat)abstract
- It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.
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| 2. |
- Gisselsson Nord, David, et al.
(författare)
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Locus-specific multifluor FISH analysis allows physical characterization of complex chromosome abnormalities in neoplasia
- 2000
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Ingår i: Genes, Chromosomes and Cancer. - 1045-2257. ; 28:3, s. 347-352
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Tidskriftsartikel (refereegranskat)abstract
- Novel techniques in molecular cytogenetics have radically improved the ability to characterize genetic changes in neoplastic cells. In parallel, a rapid development in high-throughput genomics has resulted in detailed physical maps of the human genome. Combining these two fields, we have developed a method for the simultaneous visualization of several physically defined segments along a chromosome. Seven YAC clones and one subtelomeric cosmid clone from chromosome 12 were labeled with unique combinations of four fluors and hybridized to metaphase chromosomes from neoplastic cells. In a uterine leiomyoma and a myxoid liposarcoma with translocations 12;14 and 12;16, the breakpoints in chromosome 12 could be localized to the HMGIC and CHOP regions, respectively. In the other tumors, more complex aberrations were visualized, including two inversions in 12q with a common breakpoint between MDM2 and D12S332 in a pleomorphic adenoma, amplification of MDM2 and CDK4 in ring chromosomes from a malignant fibrous histiocytoma, and amplification of KRAS2 together with other unbalanced rearrangements in two pancreatic adenocarcinomas. Combinatorially labeled single-copy probes may thus simultaneously provide physical localization of breakpoints and an overview of complex structural rearrangements.
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| 3. |
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| 4. |
- Gorunova, Ludmila, et al.
(författare)
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Extensive cytogenetic heterogeneity in a benign retroperitoneal schwannoma
- 2001
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Ingår i: Cancer Genetics and Cytogenetics. - 0165-4608. ; 127:2, s. 148-154
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Tidskriftsartikel (refereegranskat)abstract
- A benign retroperitoneal schwannoma from a patient without prior exposure to radiotherapy or chemotherapy was analyzed by chromosome banding after short-term culture. An extensive intratumor heterogeneity in the form of 29 karyotypically related as well as unrelated clones was found. The aberrant clones were diploid or near-diploid and displayed both numerical and structural changes. All chromosomes, except 11, 16, and 20, were affected. Numerical changes included trisomies X, 7, 9, 17, and 18, and monosomies 13 and 18. No clonal loss of chromosome 22, the most characteristic abnormality in schwannomas of other locations, was, however, detected. The structural aberrations resulted in a total of 58 chromosomal breakpoints, with chromosomes 18, 1, and 15 participating in rearrangements most frequently, followed by chromosomes 14, 2, and 22. A striking finding was the clonal involvement of 18p11 in eight rearrangements affecting different chromosomes, suggesting alteration of telomeric function. The molecular mechanisms underlying the observed massive polyclonality in the schwannoma, particularly the presence of cytogenetically unrelated clones, are unknown and probably heterogeneous.
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| 5. |
- Heidenblad, Markus, et al.
(författare)
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Detailed genomic mapping and expression analyses of 12p amplifications in pancreatic carcinomas reveal a 3.5-Mb target region for amplification.
- 2002
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Ingår i: Genes, Chromosomes and Cancer. - : Wiley. - 1045-2257. ; 34:2, s. 211-223
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Tidskriftsartikel (refereegranskat)abstract
- Previous cytogenetic and comparative genomic hybridization (CGH) analyses have shown that the gain of chromosome arm 12p is frequent in pancreatic carcinomas. We investigated 15 pancreatic carcinoma cell lines using CGH, fluorescence in situ hybridization (FISH), and semiquantitative polymerase chain reaction (PCR) to characterize 12p amplifications in detail. The CGH analysis revealed gains of 12p in four of the cell lines and local amplification within 12p11-12 in six cell lines. By FISH analysis, using precisely mapped YAC clones, the commonly amplified region was found to be approximately 5 Mb. The amplified segment extended from YAC 753f12, covering the KRAS2 locus, to YAC 891f1, close to the centromere. A semiquantitative PCR methodology was used to estimate genomic copy numbers of 14 precisely mapped expressed sequence tags (ESTs) and sequence-tagged sites, located within this interval. The level of amplification ranged from two- to 12-fold. The produced gene copy profiles revealed a 3.5-Mb segment with various local amplifications. This region includes KRAS2 and ranges from D12S1617 to sts-N38796. Two of the cell lines (primary and metastatic tumor from the same patient) showed amplification peaks within the distal region of this segment, two had peaks within the proximal region, one showed subpeaks in both regions, and one displayed amplification of the entire region. Chromosome segment-specific cDNA array analysis of 29 expressed sequences within the whole interval between D12S1617 and sts-N38796 indicated overexpression of four ESTs, two corresponding to DEC2 and PPFIBP1, and two to ESTs with unknown function. Expression analysis of these and of KRAS2 showed specific overexpression in the six cell lines with local 12p amplifications. These findings indicate two target regions within the 3.5-Mb segment in 12p11-12, one proximal including PPFIBP1, and one distal including KRAS2.
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| 7. |
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| 8. |
- Jonson, Tord, et al.
(författare)
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Altered expression of TGFB receptors and mitogenic effects of TGFB in pancreatic carcinomas
- 2001
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Ingår i: International Journal of Oncology. - 1019-6439. ; 19:1, s. 71-81
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Tidskriftsartikel (refereegranskat)abstract
- Alteration of the transforming growth factor beta (TGFB) signalling pathway is important in pancreatic carcinogenesis, as shown by the frequent inactivation of the downstream target SMAD4. We recently analysed a series of pancreatic carcinoma cell lines with respect to alterations of five SMAD genes involved in TGFB signalling, and showed that SMAD4 was structurally rearranged in 42% of these. This pathway may, however, also be affected by alterations of genes whose products regulate the activation of TGFB as well as of TGFB receptor genes. We therefore studied the expression of UPA, UPAR, IGF2R, ALK5 (TGFBR1), TGFBR2, TGFBR3, ENG, ALK1, TGFB1, TGFB2, and TGFB3 in a series of 14 pancreatic carcinoma cell lines. We also analysed ALK5 and TGFBR2 for mutations, cell surface localisation of TGFBR2 and ENG, and TGFB1 response. No mutations of ALK5 or TGFBR2 were found. However, 4 cell lines were methylated within the ALK5 promoter region. ALK5 expression was strongly reduced in 9 cases, whereas TGFBR2 expression was increased in 12 of the cell lines. The TGFB signalling associated receptors ENG and ALK1 were co-expressed in 4 of the cell lines. There was no evidence for disruption of the UPAR-IGF2R TGFB activating pathway. The response to TGFB1 was analysed in 12 cell lines, and 6 of these (50%) showed increased proliferation. The cell lines stimulated by TGFB showed frequent mutations of SMAD4, KRAS2, and TP53, as well as frequent absence of CDKN2B expression. These results suggest that the ALK5-SMAD4 part of the TGFB signalling pathway is a major target for inactivation in pancreatic carcinomas, that the expression of TGFBR2, TGFBR3, and receptors involved in TGFB activation are maintained, and that alterations of components of the TGFB signalling pathway may be accompanied by a positive effect of TGFB on cell growth.
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| 9. |
- Jonson, Tord, et al.
(författare)
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Characterization of genomically amplified segments using PCR: optimizing relative-PCR for reliable and simple gene expression and gene copy analyses
- 2000
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Ingår i: Genes, Chromosomes and Cancer. - 1045-2257. ; 29:2, s. 192-199
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Tidskriftsartikel (refereegranskat)abstract
- Gene amplification is one of the mechanisms for oncogene activation in solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta-actin (ACTB) and L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions.
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| 10. |
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