The CEBPA gene is targeted by the t(14;19) translocation. (A) A representative FISH image of the t(14;19)(q32;q13) in patient P5. The RP11-270I13 probe produces a red signal on the normal chromosome 19 and a split signal between der(14) and der(19). A green signal from the IGH constant region-specific probe14 identifies chromosomes 14 and der(14). Images were visualized under a Leica DM RXA microscope equipped with a fluorescence epi-illumination 100×/130-0.60 oil-immersion objective lens (Leica, Rueil-Malmaison, France). Leica QFISH software was used to digitally acquire images after capturing them with a Photometrics Sensys camera (Roper Scientific, Evry, France). (B) Quantitative RT-PCR analysis of CEBPA expression in t(14;19) patients (P1-P6), 3 control patients with BCP-ALL and the human cell lines U937 (AML) and HEP3B (hepatocellular carcinoma) using gene expression assay no. Hs.00263372_s1 (Applied Biosystems, Foster City, CA). Data are presented as percentage of ABL expression. Note that for patient 1 the bar graph is not drawn to scale: the real value is 6672%. Comparable results were obtained when the GUS gene was used as a reference. Because CEBPA is composed of a single exon, control experiments were performed with omission of the reverse transcriptase from the reaction. The observed Ct values in control experiments were always several cycles higher than in the test experiments. Quantitative RT-PCR analyses of the neighboring genes (CEBPG, PEPD, and LPR3) are shown in Figure S1. P1-P6 indicates t(14;19) samples; T1-T3, control BCP-ALL samples without a chromosome 19 abnormality. (C) Nucleotide sequence alignments of fusion CEBPA-Cmu transcripts isolated from patients P1 and P2: chromosome 19 sequences are indicated in uppercase; chromosome 14 sequences, lowercase. The JH segment is underlined on the germline chromosome 14 sequences and was identified as JH4 for P1 and JH6 for P2. The first exon of IGH constant (Cmu) gene is indicated in bold. Nucleotides underlined in P2 sequences differ from the genomic germline sequences used for comparison. (D) Western blot analyses of 200 μg protein extracted from blast cells of patients P2, P5, and P6. Proteins were separated on a 12% denaturating acrylamide gel, and transferred onto a nylon membrane. Proteins were detected using a goat anti-CEBPA immuneserum (sc 9314; Santa Cruz Biotechnology, Santa Cruz, CA). U937 and MOLT-4 (T-ALL cell line) extracts were used as positive and negative controls, respectively. Arrows indicate the p42 and p30 CEBPA protein species.
