Quantitation of bcl-2 oncogene in cultured lymphoma/leukemia cell lines and in primary leukemia B-cells by a highly sensitive RT-PCR method.
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.
Quattrone
A
A
Institute of General Pathology, University of Florence, Italy.
Papucci
L
L
Santini
V
V
Schiavone
N
N
Noferini
D
D
Calastretti
A
A
Copreni
E
E
Morelli
S
S
Rossi Ferrini
P L
PL
Nicolin
A
A
D001483:Base Sequence; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D006801:Humans; D015448:Leukemia, B-Cell / Q000235:genetics* / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D009857:Oncogenes; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D012194:RNA-Directed DNA Polymerase