Multiplex fluorescence in situ hybridization and cross species color banding of a case of chronic myeloid leukemia in blastic crisis with a complex Philadelphia translocation.

Exciting new techniques in molecular cytogenetics--namely, spectral karyotyping, multiplex fluorescence in situ hybridization (M-FISH), and cross species color banding--have been recently developed. An increasing number of reports demonstrate the success of these procedures in providing additional cytogenetic information--identifying marker chromosomes and revealing the presence of previously undetected chromosomal changes. However, these procedures have their limitations, and their absolute sensitivity in the accurate identification of subtle chromosomal abnormalities remains to be established. M-FISH and color banding have been applied to a case of chronic myeloid leukemia with a complex Philadelphia translocation involving chromosomes 9, 17, and 22, which had initially been identified from G-banded chromosome analysis. The abnormalities were confirmed by chromosome "painting" and specific probes. Although M-FISH and color banding revealed no additional cryptic chromosomal changes, this study has clearly demonstrated the success of these multiple color FISH approaches in the accurate characterization of a complex rearrangement with subtle abnormalities.

Characterization of the human myeloid leukemia-derived cell line GF-D8 by multiplex fluorescence in situ hybridization, subtelomeric probes, and comparative genomic hybridization.

The human myeloid leukemia cell line GF-D8 was established from the peripheral blood blasts of a patient with acute myeloid leukemia FAB subtype MI (AML-MI). The karyotype, which has not changed significantly over several years of culture, was described initially as 44,XY,-5,del(7q),inv(7q),add(8q),add(11q),del(12p),-15,-17,+mar. With the advent of multicolor fluorescence in situ hybridization (FISH) techniques, the prospect of accurately characterizing this complex karyotype became feasible. In the present study, we applied 24-color whole-chromosome painting and analyzed the results using a filter-based detection system and proprietary software for multiplex FISH (M-FISH). This resulted in the refinement of the karyotype and the identification of hitherto unsuspected chromosome rearrangements. M-FISH identified the origin of the add(8q) and add(11q) as well as the small marker chromosome. Both the del(7q) and del(12p) were redefined as unbalanced translocations and an apparently normal chromosome 11 was shown to be t(11;17). Importantly, the del(12p) was shown to be a der(12)t(7;12). Single-color whole-chromosome painting studies confirmed these findings, but also identified a cryptic t(Y;12) not seen in the original M-FISH analysis. We then carried out a FISH screening assay using a complete set of chromosome-specific subtelomeric probes. This allowed the identification of p and q subtelomeric regions involved in the translocations and indicated amplification of the 8q subtelomeric region. Comparative genomic hybridization (CGH) revealed a highly unbalanced karyotype, as deletions accompanied the majority of translocations, and identified the regions of amplification as 8q22.3-qter and 11q21-qter. Finally, conventional FISH with centromeric and unique sequence probes was necessary to elucidate all of the rearrangements.

Transformation of rat tracheal epithelial cells to immortal growth variants by particulate and soluble nickel compounds.

The cytotoxicity and transforming activity of nickel subsulfide, nickel oxide and nickel sulfate was studied by assays of colony-forming efficiency and of transformation of rat tracheal epithelial (RTE) cells to enhanced growth variants (EGVs) and immortal growth variants (IGVs). Nickel subsulfide caused dose-dependent cytotoxicity between 1 and 5 micrograms/ml, whereas the cytotoxic range of nickel oxide and nickel sulfate was 50-200 micrograms/ml and 60-130 micrograms/ml, respectively. At lower concentrations, nickel sulfate caused modest (up to 126%) growth stimulation. During the initial 24-h treatment period, internalized nickel subsulfide particles were observed in phagocytic vesicles in cells near the periphery of all RTE cell colonies, whereas nickel oxide particles were not internalized but had adhered to both the cells and the tissue culture dish. After 7-10 days of the transformation assay, nickel subsulfide particles were no longer visible, but nickel oxide particles remained on the dish for the duration of the 5 week assay. During weeks 3-5 of the transformation assay, internalized nickel oxide particles were observed in non-vacuolated cells at the periphery of the colonies. All 3 nickel compounds significantly (p < 0.05) increased the transformation frequency of RTE cells to EGVs at moderately cytotoxic concentrations; the order of potency was Ni3S2 > NiO = NiSO4. MNNG, the positive control, was twice as active as nickel subsulfide at 1/3 the concentration and 1/6 the duration of treatment. EGVs induced by MNNG, nickel subsulfide and nickel sulfate were cloned and converted to IGVs at frequencies of 44, 24 and 43%, respectively. In contrast, EGVs transformed by nickel oxide rarely converted to IGVs (13%). All nickel-induced IGVs were immunohistochemically epithelial, mitotically active, aneuploid and exhibited high plating efficiencies. Our results suggest that respiratory epithelial cells are targets for the transforming capabilities of several nickel compounds but that the potency and mechanism of transformation by various forms of nickel may be different according to the physico-chemical properties of each compound.