Comparative M-FISH and CGH analyses in sensitive and drug-resistant human T-cell acute leukemia cell lines

Abstract

Cell lines of human T-cell acute lymphoblastic leukemias (T-ALL) have gained high interest for study of mechanisms of cytostatic drug resistance. However, they should also be suited to examine the validity and reliability of molecular cytogenetic techniques in detecting genomic alterations in neoplastic cells. Therefore, comparative genomic hybridization (CGH) and 24-color-fluorescence-in-situ-hybridization (M-FISH) were applied to eight sublines of CCRF-CEM leukemia cells selected in vitro for drug resistance and to their drug-sensitive parental counterparts. All cell lines were characterized by altered chromosome numbers and by a variety of chromosomal structural aberrations as shown by M-FISH. The great majority of anomalies detected by this technique were confirmed by CGH. Interestingly, a considerable number of the rearrangements found were imbalanced. Amplifications of 5q13 in the six methotrexate-resistant cell lines, a del(9)(p21pter) in all lines examined, and a gain of chromosome 20 in 9 of the 10 lines examined were readily detected by both techniques. The same held true for losses of chromosomes 17 and 18 in the near tetraploid cell lines which could also be confirmed by CGH. Some imbalances of genomic material detected by CGH were, however, not observed by means of M-FISH, possibly due to the limited extension of the corresponding chromosomal segment involved or the small subpopulation of cells affected. On the other hand, reciprocal translocations, balanced isochromosomes, and small deletions remained mainly undetected by CGH. A comparison of chromosomal alterations in drug-resistant and parental cell lines showed not only amplifications of chromosomal segments harboring well-known drug resistance genes, e.g., the dihydrofolate reductase gene, but also chromosomal changes which may involve novel genes associated with drug resistance. Thus, the present study has clearly unveiled the strengths and weaknesses of both techniques which can excellently complement each other. Their combination allowed a distinct improvement of the definition of the complex karyotypes of drug-resistant cell lines.

FISH studies on the telomeric regions of the T-cell acute lymphoblastic leukemia cell line CCRF-CEM

“…Since that time, new and apparently rather stable marker chromosomes arose in the karyotype along evolution in vitro (Pittman et al, 1993). Recently multicolor-FISH in combination with comparative genomic hybridization (CGH) defined der(5)t(5;15), del(8)(pter), del(9) (pterp13), der(9)t(8;9), and der(15)t(5;15), as markers of this cell line (Weise et al, 2002).…”

“…The CCRF-CEM cell line, which was originally derived from a T-cell acute lymphoblastic leukemia (Foley et al, 1965) has been karyotypically analyzed by classic chromosome G-banding (Moore et al, 1985;Pittman et al, 1993), by comparative genomic hybridization and by multicolor-FISH (Weise et al, 2002). Its chromosome number is 42-47.…”

“…Cell lines characterized by defined marker chromosomes should be a good model to study this aspect. We have examined the telomeres of a karyotypically rather well characterized Tcell acute lymphoblastic leukemia (T-ALL) cell line (CCRF-CEM) with several defined markers (Weise et al, 2002) to compare the telomere lengths of these markers with those of their chromosomes of origin. Peptide nucleic acid (PNA) probes were applied for FISH detection of telomeres and quantitative estimation of their relative lengths, as these probes have been shown to be highly specific and most efficient for this purpose (Lansdorp et al, 1996;Martens et al, 1998;Perner et al, 2003).…”

Cytogenetic characterisation and proteomic profiling of the Imatinib-resistant cell line KCL22-R

“…Multiplex-FISH (M-FISH) was performed to confirm GTG-results as described by Weise and co-workers (25). Ten metaphase images per cell line were captured on a fluorescence microscope (Axioplan 2, Zeiss, Germany) with a PCO VC45 CCD camera (PCO, Kehl, Germany) and suitable filter combinations (DAPI/FITC/Spectrum Orange/Texas Red/ Cyanine 5/Cyanine 5.5) using ISIS3-software (MetaSystems, Altlussheim, Germany).…”

“…Ten metaphase images per cell line were captured on a fluorescence microscope (Axioplan 2, Zeiss, Germany) with a PCO VC45 CCD camera (PCO, Kehl, Germany) and suitable filter combinations (DAPI/FITC/Spectrum Orange/Texas Red/ Cyanine 5/Cyanine 5.5) using ISIS3-software (MetaSystems, Altlussheim, Germany). 24-Color-FISH using all 24 human whole chromosome painting probes was applied to identify interchromosomal rearrangements (25).…”