SENSITIVITY TO FAS-MEDIATED APOPTOSIS IN PEDIATRIC ACUTE LYMPHOBLASTIC-LEUKEMIA IS ASSOCIATED WITH A MUTANT P53 PHENOTYPE AND ABSENCE OF BCL-2 EXPRESSION

Fas (APO-1/CD95) is a cell-surface protein that can mediate apoptosis upon specific ligand or antibody binding. The Bcl-2 protein may functi on as a modulator of Fas-induced apoptosis by blocking a downstream ac tivation step, and Bcl-2 expression in acute lymphoblastic leukemia (A LL) cells appears to depend partly on expression of a wild-type (wt) p 53 tumor suppressor gene (Findley ef al, Blood 1997; 89: 2986). We the refore investigated the relationship between sensitivity to Fas-mediat ed apoptosis and (1) Fas expression, (2) p53 status, and (3) Bcl-2 pro tein levels in pediatric ALL cell lines and primary leukemic cells. Ce ll lines included 21 B cell precursor (BCP)-ALL and four T-ALL lines; in five cases, cryopreserved primary leukemic cells from which these l ines were established were also examined. Additionally, we evaluated t he effect of anti-fas monoclonal antibody on the activation of proteas e CPP32 and induction of apoptosis in these lines. By SSCP analysis an d DNA sequencing, we detected p53 mutations (mt) in eight out of 25 AL L cell lines (exon-7, codon 248 n = 6; exon-8, codon 273, n = 2). The expression of Fas and Bcl-2 was examined by immunofluorescence stainin g and quantified as the number of molecules of equivalent soluble fluo rochrome (MESF), Elevated levels of Fas were expressed in all six line s with a mutation of p53 in codon 248 (1500 to 10 800 MESF). Although Fas was detectable in seven of the 17 lines with wt-p53, expression wa s lower (150-900 MESF) compared with mt-p53+ lines. Bcl-2 was expresse d in PO of the 25 lines. Most (9/10) wt-p53+ lines expressed Bcl-2, wh ereas only one of eight mt-p53+ lines and no p53-null lines expressed this protein. Treatment of Fas-positive lines with anti-fas monoclonal antibody (200 ng/ml) for 6 h induced activation of CPP32 and apoptosi s In eight of 13 Fas+ lines. Sensitivity to Fas-mediated apoptosis was associated with a mt-p53 phenotype and absence of Bcl-2 expression, S ix of eight Fas+/Fas-sensitive (S) lines were mt-53+/Bcl-2-, whereas o nly two Fas+/Fas-S lines were wt-p53+/Bcl-2+; both of these latter lin es expressed low levels of Bcl-2 compared to Fas-resistant lines. In c ontrast, four of five Fas+/Fas-resistant (R) lines were wt-p53+/Bcl-2; the exception was p53-null/Bcl-2- but expressed a low level of Fas ( 150 MESF). Activation of the cysteine protease CPP32 and cleavage of i ts substrate poly(ADP-ribose)polymerase (PARP) was also detected in Fa s-S but not Fas-R lines. We obtained similar results from both the pri mary leukemic cells and the corresponding cell lines in five cases: ov erexpression of Fas and Fas-sensitivity were present In mt-p53+/Bcl-2- but not wt-p53+/Bcl-2+ cells. These results suggest that some pediatr ic ALL cells expressing mt-p53+ may be sensitive to Fas-mediated apopt osis due to high levels of Fas expression and lack of Bcl-2, and furth er suggest that molecular methods of activating Fas may be useful for therapy of refractory ALL with the Fas+/mt-p53+ phenotype.