Quantitation of bcl-2 oncogene in cultured lymphoma/leukemia cell lines and in primary leukemia B-cells by a highly sensitive RT-PCR method

Abstract

BACKGROUND. The bcl-2 gene, isolated from the t(14;18) chromosomal translocation breakpoint, is able to prevent apoptotic death induced by various stimuli in different tissues. Therefore bcl-2 oncogene expression could be a key parameter for investigating the molecular mechanisms involved in the apoptosis of normal and neoplastic hematopoietic cells. METHODS. In order to evaluate bcl-2 expression in both follicular B-lymphomas carrying or not carrying the 14;18 translocation and in lymphatic leukemias, we optimized an internal standard-based method of reverse transcriptase-polymerase chain reaction (RT-PCR) for the rapid quantitation of bcl-2 mRNA cellular levels. A simple purification of the reverse transcription products resulted in very high PCR efficiency, so that radioactive labelling of the amplification products was avoided. RESULTS. bcl-2 mRNA levels proved to be higher in t(14;18) than in t(14;18) negative cell lines, and higher in primary leukemia pre-B cells than in early-B cells. Tested for sensitivity by identifying minimal residual t(14;18) B cells expressing the bcl-2/IgH gene, this RT-PCR method was able to detect bcl-2/IgH mRNA from just one t(14;18) positive cell out of ten million t(14;18) negative cells. CONCLUSIONS. The RT-PCR method we optimized appears to be suitable for clinical use in both leukemia/lymphoma characterization and in lymphomatous disease follow-up.