Chronic rnyelogenous leukemia (CML) is a stem cell disorder characterized by the Philadelphia (Ph) chromosome which generates the fusion message BCR-ABL and the fusion protein p210. In addition, CML patients express a smaller p190 BCR-ABL transcript encoding a 190 kD protein. It was the aim of the present study to investigate p!90 mRNA expression in highly purified CD34+, CD34 and CD15+ cells enriched by magnetic cell sorting (MACS) eventually combined with fluorescent-activated cell sorting (FACS). Cell fractions were purified from either bone marrow (BM) or peripheral blood (PB) of 8 CML patients at diagnosis. CD34cells were 100% Ph chromosome positive at standard cytogenetic analysis. Comparable numbers of each cell fraction (105, IxlO6, 2x10) were analyzed for the presence of BCR/ABL transcripts (p210, p!90) by reverse transcription polymerase chain reaction (RT-PCR). All samples [CD34(n = 8); CD34 (n = 7); CD15+ (n = 7)] were p210 positive. By means of a quantitative assay, p210 BCR-ABL expression was compared in paired CD34+ and CD15+ samples (n = 4). Such a quantitative analysis revealed a 2- to 10-fold decrease of the number of transcripts per -4g of RNA in CD15+ cells as compared (o CD34+ cells. All CD34+ fractions expressed the p!90 transcript. In striking contrast, p!90 transcript was inconsistently detected in CD34 (3 out of 7 samples) and CD 15+ (2 out of 7 samples) cells. To further investigate the relationship between p 190 expression and cell maturation, CD34+ cells (1x106) were cultured in suspension for 14 days in the presence of SCF (50 ng/ml), IL-3 (50 ng/ml), G-CSF (100 ng/ml) and IL-6 (50 ng/ml). RT-PCR analysis of cultured cells (CD34' = 0%; CDllb'CDlS+ = >75%) failed to reveal p!90 mRNA expression. In conclusion, our data demonstrate that: (i) in addition to p210 transcript, CD34+ cells enriched from CML at diagnosis express p!90 BCR-ABL; (ii) both p210 and p190 transcripts are downregulated during cell maturation; (iii) monitoring p190 BCR-ABL expression in immature and mature cell fractions might be of pathophysiological and therapeutical relevance.
Expression of P190 and P2IO BCR-ABL in CML-derived CD34+, CD34-and GDIS' cells
Carlo Stella C.;
1998-01-01
Abstract
Chronic rnyelogenous leukemia (CML) is a stem cell disorder characterized by the Philadelphia (Ph) chromosome which generates the fusion message BCR-ABL and the fusion protein p210. In addition, CML patients express a smaller p190 BCR-ABL transcript encoding a 190 kD protein. It was the aim of the present study to investigate p!90 mRNA expression in highly purified CD34+, CD34 and CD15+ cells enriched by magnetic cell sorting (MACS) eventually combined with fluorescent-activated cell sorting (FACS). Cell fractions were purified from either bone marrow (BM) or peripheral blood (PB) of 8 CML patients at diagnosis. CD34cells were 100% Ph chromosome positive at standard cytogenetic analysis. Comparable numbers of each cell fraction (105, IxlO6, 2x10) were analyzed for the presence of BCR/ABL transcripts (p210, p!90) by reverse transcription polymerase chain reaction (RT-PCR). All samples [CD34(n = 8); CD34 (n = 7); CD15+ (n = 7)] were p210 positive. By means of a quantitative assay, p210 BCR-ABL expression was compared in paired CD34+ and CD15+ samples (n = 4). Such a quantitative analysis revealed a 2- to 10-fold decrease of the number of transcripts per -4g of RNA in CD15+ cells as compared (o CD34+ cells. All CD34+ fractions expressed the p!90 transcript. In striking contrast, p!90 transcript was inconsistently detected in CD34 (3 out of 7 samples) and CD 15+ (2 out of 7 samples) cells. To further investigate the relationship between p 190 expression and cell maturation, CD34+ cells (1x106) were cultured in suspension for 14 days in the presence of SCF (50 ng/ml), IL-3 (50 ng/ml), G-CSF (100 ng/ml) and IL-6 (50 ng/ml). RT-PCR analysis of cultured cells (CD34' = 0%; CDllb'CDlS+ = >75%) failed to reveal p!90 mRNA expression. In conclusion, our data demonstrate that: (i) in addition to p210 transcript, CD34+ cells enriched from CML at diagnosis express p!90 BCR-ABL; (ii) both p210 and p190 transcripts are downregulated during cell maturation; (iii) monitoring p190 BCR-ABL expression in immature and mature cell fractions might be of pathophysiological and therapeutical relevance.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
