Flow karyotyping of human melanoma cell lines

Abstract

Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.Key terms: Chromosomes, flow cytometry Flow cytometry is widely used for studying the diagnostic and prognostic significance of DNA content differences in human tumors. The advantage of this technique is that rapid ploidy determinations can be made on large numbers of cells. However, the information provided by measuring total DNA content gives only a rather crude estimate for numerical chromosome abnormalities. Potentially, more detailed information can be obtained by flow cytometric measurement of the DNA content of isolated chromosomes. The evolution of this technique during the last 5 years has now made it possible to identify a variety of chromosomal polymorphisms and abnormalities and to sort individual chromosomes for gene mapping and cloning experiments (13,201. So far, only few reports on the application of flow cytometry for karyotyping of solid tumors have appeared (11,141.The purpose of the present study was to explore the possibility of using flow karyotyping for characterizing genetic differences between seven different human melanoma cell lines. We chose this neoplasm because melanoma shows a high degree of genetic instability, and cell lines from this tumor can be relatively easily maintained in tissue culture. The variation in ploidy level of the melanoma cell lines was determined by chromosome Table 1. Human foreskin fibroblasts (VH-25, 10th passage) were kindly provided by Dr. P. Abraham, Department of Medical Biochemistry, University Medical Center, Leiden. The cell lines were maintained in continuous cultures in 75-cm3 plastic culture flasks on Ham's F-12 medium supplemented with 10% foetal calf serum and antibiotics and passaged twice a week.Chromosome Isolation Confluent cultures were trypsinized and plated out in a 140-4 ratio into new culture flasks. After reaching 75% confluency (usually in about 48 h) colchicin (Colcemid, Gibco, Madison, WI) was added to the culture medium to a final concentration of 0.05 hg/ml. Mitotic cells were harvested 15-18 h later by gently tapping the side of the culture flask. Next the cells were centrifuged at 175g for 5 min at room temperature, and the pellet was washed once with hypotonic KC1 solution (0.075 M). After that, the c...

Clinical applications of flow karyotyping in myelocytic leukemia by stimulation of different subpopulations of cells in blood or bone marrow samples

“…Flow karyotyping is a well-established method of analyzing chromosomes in suspension by means of a flow cytometer, resulting in either uni-or bivariate flow karyograms. In these karyograms virtually all human chromosomes can be discriminated on the basis of their size and base pair ratio (1)(2)(3)9,10,13,17,18). The resulting chromosome clustering pattern is characteristic for the species from which the chromosomes are derived.…”

Flow Cytogenetics

“…Flow karyotype analysis of cell lines established from patients with Burkitt's lymphoma [17] and melanoma [19] was successful in detecting clinically important structural and numerical chromosome aberrations. The typical 14q+ chromosome in the t(8; 14) case, and two 'variant translocations', the 2q+ in the t(2;8) and the small 22q~ in the t(8;22) cases, could be identified as single peaks within the flow karyotypes of several differ ent Burkitt lymphoma lines [17].…”