The retinoic acid (RA)-sensitive NB4 cell line was the first established acute promyelocytic leukemia (APL) permanent cell line. It harbors the (15;17) translocation, which fuses the PML and RARA genes. Given the low frequency of APLs, their generally low white blood cell count, and the difficulty to work on APL patient cells, this cell line represents a remarkable tool for biomolecular studies. To investigate possible mechanisms of retinoid resistance, subclones of NB4 resistant to all-trans retinoic acid (ATRA) were established. To characterize better the parental NB4 cell line and four ATRA-resistant subclones (NB4-R4, NB4-A1, NB4-B1, and NB4-007/6), we have performed both conventional and 24-color FISH karyotyping. Thus, we could identify all chromosomal abnormalities including marker chromosomes that were unclassified with R banding. Moreover, we have performed dual-color FISH by use of specific PML and RARA probes, to evaluate the number of copies for each gene and fusion gene. Interestingly, the number of copies of PML, RARA, and fusion genes was different for each cell line. Finally, we assessed the presence of the PML, RARA, PML/RARA, and RARA/PML transcripts by RT-PCR and of the PML/RARA and RARA proteins by Western blotting in all the cell lines. These data could focus further research for a better understanding of the molecular mechanisms underlying response or resistance to differentiating and/or apoptotic reagents.

Molecular cytogenetics of the acute promyelocytic leukemia-derived cell line NB4 and of four all-trans retinoic acid-resistant subclones

FANELLI, MIRCO;
2002-01-01

Abstract

The retinoic acid (RA)-sensitive NB4 cell line was the first established acute promyelocytic leukemia (APL) permanent cell line. It harbors the (15;17) translocation, which fuses the PML and RARA genes. Given the low frequency of APLs, their generally low white blood cell count, and the difficulty to work on APL patient cells, this cell line represents a remarkable tool for biomolecular studies. To investigate possible mechanisms of retinoid resistance, subclones of NB4 resistant to all-trans retinoic acid (ATRA) were established. To characterize better the parental NB4 cell line and four ATRA-resistant subclones (NB4-R4, NB4-A1, NB4-B1, and NB4-007/6), we have performed both conventional and 24-color FISH karyotyping. Thus, we could identify all chromosomal abnormalities including marker chromosomes that were unclassified with R banding. Moreover, we have performed dual-color FISH by use of specific PML and RARA probes, to evaluate the number of copies for each gene and fusion gene. Interestingly, the number of copies of PML, RARA, and fusion genes was different for each cell line. Finally, we assessed the presence of the PML, RARA, PML/RARA, and RARA/PML transcripts by RT-PCR and of the PML/RARA and RARA proteins by Western blotting in all the cell lines. These data could focus further research for a better understanding of the molecular mechanisms underlying response or resistance to differentiating and/or apoptotic reagents.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2506627
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