pq-COBRA-FISH and premature chromosome condensation; a novel combination in molecular cytogenetics

Human chromosomes have been studied for over a century, but it took until the 1950s that the correct human chromosome number was determined. The development of chromosome pretreatment procedures and modification of DNA staining techniques some 20 years later, allowed more precise chromosome identification based on banding patterns. This landmarkadvance in human cytogenetics allowed researchers to address specific clinical and research questions and make rapid progress in understanding the chromosomal basis of many genetic diseases and cancer. Although banding methodologies allow the distinction of individual chromosome pairs and the detection of gross karyotype aberrations, the analysis is heavily dependent on the accumulated experience and sometimes subjective interpretation of cytogeneticists. The low mitotic index, poor growth rate and sub-optimal chromosome morphology, which are hallmarks of tumour metaphase chromosome preparations, make cancer cytogenetics a particularly difficult discipline. Introduction of fluorescent in situ hybridisation (FISH) in combination with recombinant DNA technology opened a new chapter in detection and investigation of chromosomal basis of human genetic diseases in the 1990s. The success of this new discipline, molecular cytogenetics, in gene mapping and precise characterisation of chromosomal abnormalities, hinged on development of nucleic acids modification to incorporate fluorescent reporter molecules into nucleic acid hybridization probes. This thesis contributes to further advancement of molecular cytogenetics. The pq-COBRAFISH method presented in Chapter 2, employs a combination of binary and ratio-labelling to discriminate in one experiment all human chromosome arms, thus enhancing and objectifying the detection of chromosomal abnormalities. In Chapter 3, it has been used to detect chromosomal rearrangements in individuals with abnormal phenotype and normal G-banding patterns. It also aided in identification of a novel cancerous gene alteration by discovering BCL11B, as a new candidate gene in acute myelocytic leukaemia. pq-COBRA-FISH readily resolves complex karyotypes, characteristic of solid tumours. As such it was instrumental to discovery of a novel function of the chromatin remodelling complex, hSNF5, in maintenance of chromosome stability and ploidy (Chapter 4). Although molecular cytogenetics with its sophisticated multicolour FISH technology has allowed significant advancement in cancer and has become an intricate part of diagnosis and research there are still analytical and methodological problems in cancer cytogenetics. Full karyotyping, for example, is still restricted to mitotic cells. Chapter 5 of this thesis presents studies where a potent phosphatase inhibitor, calyculin A, was employed to prematurely condense the chromosomes of non-mitotic cells. This premature chromosome condensation technique or PCC increases the number of analysable chromosome structures compared to classical mitotic arrest, with minimum culture. This greatly improves the capacity of researchers to cytogenetically study solid tumours, where the success of comprehensive karyotyping of biopsy material has always been problematic. The specific morphological characteristics of PCC chromosomes furthermore allow distinction of the G1 and G2 phases of the cell cycle. The possibility to assess (differences in) chromosomal abnormalities of cells in the various cell cycle phases can offer valuable insight into the mechanisms underlying the tumorigenesis processes, including the much debated chromosome instability.Kreatech Diagnostics (Amsterdam, The Netherlands) Stichting Leids Universitair Fonds (LUF)Microscopische beeldvorming en technologi

pq-COBRA-FISH and premature chromosome condensation; a novel combination in molecular cytogenetics

Human chromosomes have been studied for over a century, but it took until the 1950s that the correct human chromosome number was determined. The development of chromosome pretreatment procedures and modification of DNA staining techniques some 20 years later, allowed more precise chromosome identification based on banding patterns. This landmarkadvance in human cytogenetics allowed researchers to address specific clinical and research questions and make rapid progress in understanding the chromosomal basis of many genetic diseases and cancer. Although banding methodologies allow the distinction of individual chromosome pairs and the detection of gross karyotype aberrations, the analysis is heavily dependent on the accumulated experience and sometimes subjective interpretation of cytogeneticists. The low mitotic index, poor growth rate and sub-optimal chromosome morphology, which are hallmarks of tumour metaphase chromosome preparations, make cancer cytogenetics a particularly difficult discipline. Introduction of fluorescent in situ hybridisation (FISH) in combination with recombinant DNA technology opened a new chapter in detection and investigation of chromosomal basis of human genetic diseases in the 1990s. The success of this new discipline, molecular cytogenetics, in gene mapping and precise characterisation of chromosomal abnormalities, hinged on development of nucleic acids modification to incorporate fluorescent reporter molecules into nucleic acid hybridization probes. This thesis contributes to further advancement of molecular cytogenetics. The pq-COBRAFISH method presented in Chapter 2, employs a combination of binary and ratio-labelling to discriminate in one experiment all human chromosome arms, thus enhancing and objectifying the detection of chromosomal abnormalities. In Chapter 3, it has been used to detect chromosomal rearrangements in individuals with abnormal phenotype and normal G-banding patterns. It also aided in identification of a novel cancerous gene alteration by discovering BCL11B, as a new candidate gene in acute myelocytic leukaemia. pq-COBRA-FISH readily resolves complex karyotypes, characteristic of solid tumours. As such it was instrumental to discovery of a novel function of the chromatin remodelling complex, hSNF5, in maintenance of chromosome stability and ploidy (Chapter 4). Although molecular cytogenetics with its sophisticated multicolour FISH technology has allowed significant advancement in cancer and has become an intricate part of diagnosis and research there are still analytical and methodological problems in cancer cytogenetics. Full karyotyping, for example, is still restricted to mitotic cells. Chapter 5 of this thesis presents studies where a potent phosphatase inhibitor, calyculin A, was employed to prematurely condense the chromosomes of non-mitotic cells. This premature chromosome condensation technique or PCC increases the number of analysable chromosome structures compared to classical mitotic arrest, with minimum culture. This greatly improves the capacity of researchers to cytogenetically study solid tumours, where the success of comprehensive karyotyping of biopsy material has always been problematic. The specific morphological characteristics of PCC chromosomes furthermore allow distinction of the G1 and G2 phases of the cell cycle. The possibility to assess (differences in) chromosomal abnormalities of cells in the various cell cycle phases can offer valuable insight into the mechanisms underlying the tumorigenesis processes, including the much debated chromosome instability.</p

PCC and COBRA-FISH a new tool to characterize primary cervical carcinomas: To assess hall-marks and stage specificity

A newly developed assay based on chemically induced premature chromosome condensation (PCC) and multi-color combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) techniques have been implemented in order to investigate for the first time for recurrent cytogenetic aberrations in primary cervical carcinoma (derived directly from biopsies) at different stages of progression. The cytogenetic profiles of 17 biopsies derived from 14 and 3 cervical cancer patients with squamous-cell carcinomas (Sq) and with adenocarcinomas (Ad), respectively, were assessed. Frequencies of both structural as well as numerical aberrations were found to be higher in Sq than in Ad. The analysis revealed that even in early tumors stages (IB1) have a higher frequency of chromosome-losses and -gains as well as chromosomal alterations as compared to normal cells. A positive trend was found between stage advancement of cervical tumors and the frequency of numerical and structural aberrations. No specific and common chromosomal abnormality (e.g. distinct clones of translocation) was found among cervical carcinoma at the different stages (IB1, IIA and IIB). However, a distinct difference was found between stage IIIB and lower tumor stages, as all analyzed IIIB samples revealed a near tetraploid karyotype. Furthermore, all studied metaphases were aberrant and had a high frequency of translocations. PCC-COBRA-FISH characterization of a common type of an established culture from cervical carcinoma CSCC-1 revealed a triploidy/tetraploidy karyotype with several structural aberrations. In general, no similarity was found between this model and early stages of primary tumors. The newly established assay has a novel potential and can reveal the original status of primary tumors at different stages. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.Toxicogenomics and risk assessmen

PCC and COBRA-FISH a new tool to characterize primary cervical carcinomas: To assess hall-marks and stage specificity

A newly developed assay based on chemically induced premature chromosome condensation (PCC) and multi-color combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) techniques have been implemented in order to investigate for the first time for recurrent cytogenetic aberrations in primary cervical carcinoma (derived directly from biopsies) at different stages of progression. The cytogenetic profiles of 17 biopsies derived from 14 and 3 cervical cancer patients with squamous-cell carcinomas (Sq) and with adenocarcinomas (Ad), respectively, were assessed. Frequencies of both structural as well as numerical aberrations were found to be higher in Sq than in Ad. The analysis revealed that even in early tumors stages (IB1) have a higher frequency of chromosome-losses and -gains as well as chromosomal alterations as compared to normal cells. A positive trend was found between stage advancement of cervical tumors and the frequency of numerical and structural aberrations. No specific and common chromosomal abnormality (e.g. distinct clones of translocation) was found among cervical carcinoma at the different stages (IB1, IIA and IIB). However, a distinct difference was found between stage IIIB and lower tumor stages, as all analyzed IIIB samples revealed a near tetraploid karyotype. Furthermore, all studied metaphases were aberrant and had a high frequency of translocations. PCC-COBRA-FISH characterization of a common type of an established culture from cervical carcinoma CSCC-1 revealed a triploidy/tetraploidy karyotype with several structural aberrations. In general, no similarity was found between this model and early stages of primary tumors. The newly established assay has a novel potential and can reveal the original status of primary tumors at different stages. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved