C. Wuchter

High Expression Levels of X-Linked Inhibitor of Apoptosis Protein and Survivin Correlate with Poor Overall Survival in Childhood de Novo Acute Myeloid Leukemia

Purpose: Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. Experimental Design: Using flow cytometry and Western blot analysis, we, therefore, investigated 45 leukemic cell samples from children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins [CD95, Bcl-2, Bax, Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein–1 (cIAP-1), survivin]. Results: XIAP (P < 0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by French-American-British (FAB) morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (P < 0.01) and Bcl-xL (P < 0.01) expression was lower in patients with favorable rather than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP [30 (n = 10) versus 41 months (n = 34); P < 0.05] and survivin [27 (n = 10) versus 41 months (n = 34); P < 0.05]. Conclusions: We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups, and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics, and poor overall survival should be confirmed within prospective pediatric AML trials.

In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-κB activity

The TNF-related apoptosis-inducing ligand (TRAIL) is currently under evaluation as a possible (co-)therapeutic in cancer treatment. We therefore examined 129 cell samples from patients with de novo acute leukemia as to their constitutive susceptibility to TRAIL-induced apoptosis in vitro. Only 21 (16%) cell samples revealed at least 10% TRAIL-susceptible cells/sample as detected by flow cytometric annexinV staining after 24u2009h culture compared with medium control. Precursor B cell ALL samples (11 (27%) of 41) were more TRAIL-susceptible compared with AML (5 (9%) of 54; Pu2009<u20090.05) but not compared with precursor t cell all (5 (15%) of 34; Pu2009=u20090.20). Furthermore, we examined constitutive mRNA expression levels of TRAIL receptors R1–R4 by semi-quantitative RT-PCR (nu2009=u200958). Expression levels were heterogeneous, however, there was no significant correlation between the expression of the signal-transducing receptors (R1, R2) as well as of the decoy receptors (R3, R4) and TRAIL sensitivity in this series. Constitutive NF-κB activity has been shown to influence TRAIL susceptibility of leukemic cells. In 39 leukemic cell samples examined, we found a generally high NF-κB activity as detected by electrophoretic mobility shift assay which did not differ between TRAIL-susceptible and TRAIL-resistant cases. Finally, 49 acute leukemic cell samples were coincubated with doxorubicin in vitro. Doxorubicin sensitized four of 35 initially TRAIL-resistant samples and augmented TRAIL-induced apoptosis in two of 14 TRAIL-susceptible samples. In summary, constitutive TRAIL susceptibility differs between leukemia subtypes and does not correlate with mRNA expression levels of the TRAIL receptors R1–R4 as well as constitutive NF-κB activation status. The observed sensitization of leukemic cells to TRAIL by doxorubicin in vitro indicates that TRAIL should be further evaluated as to its possible role as an in vivo cotherapeutic in acute leukemia.

Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics

Resistance to chemotherapy-induced apoptosis and a multidrug-resistance (MDR) phenotype, mainly mediated by P-glycoprotein (P-gp), contribute to chemotherapy failure in hematologic malignancies. To study apoptosis-regulating factors in acute myeloid leukemia (AML), we investigated cell samples of adults with de novo AML by flow cytometry for constitutive expression levels of the apoptosis-related molecules CD95 (n = 135), Bcl-2 (n = 131), and Bax (n = 66), as well as spontaneous apoptosis in vitro (n = 104) and susceptibility to anti-CD95-induced apoptosis (CD95 sensitivity) (n = 93). We correlated these findings with P-gp function as detected by the rhodamine123-efflux test (n = 121), immunophenotype, FAB morphology, cytogenetics, and clinical data of the examined patients. Immature FAB M0/1 AML cells expressed significantly more Bcl-2 (P < 0.0002) and less cd95 (P < 0.0003) compared with aml cells of the more mature fab m2–5 subtypes. no maturation-dependent difference in bax expression was observed. fab m2–5 aml cells were more susceptible to anti-cd95-induced apoptosis (P < 0.008) and showed a lower p-gp function (P < 0.002) than fab m0/1 aml cells. leukemic cells of aml patients who achieved a complete remission (cr) after induction chemotherapy expressed less bcl-2 than non-responder (nr) (69 cr, 23 nr; P = 0.05). CR was associated with a higher extent of spontaneous apoptosis in vitro (58 CR, 17 NR; P=0.05) and a tendency towards a higher CD95 expression (73 CR, 23 NR; P = 0.08) compared to NR. CR also correlated with a low P-gp function (70 CR, 21 NR; P = 0.008) and a tendency towards CD34 negativity (73 CR, 23 NR; P = 0.08). No correlation between Bax expression and response to induction chemotherapy (49 CR, 12 NR) was observed. In stepwise logistic regression analyses, P-gp function and the extent of spontaneous apoptosis in vitro as well as CD95 sensitivity but not Bcl-2, CD95, Bax, and CD34 expression levels emerged as significant markers for response to induction chemotherapy. We conclude that the constitutive expression of CD95 and Bcl-2, as well as CD95 sensitivity and P-gp function but not constitutive Bax expression depend on the maturation stage of leukemic cells in adult de novo AML. P-gp function, the extent of spontaneous apoptosis in vitro and CD95 sensitivity are more predictive for response to induction chemotherapy in adult de novoAML than the constitutive expression levels of the apoptosis-related molecules CD95, Bcl-2 and Bax.

Correlation of protein expression and gene expression in acute leukemia

Flow cytometry (FC) is a standard method for diagnosing and subclassifying acute myeloid (AML) and acute lymphoblastic (ALL) leukemias and allows the analysis of cell surface and intracellular proteins. In the future, diagnostic procedures may include oligonucleotide microarray analysis (MA) to detect expression patterns of large numbers of specific genes.

Apoptotic response to homoharringtonine in human wt p53 leukemic cells is independent of reactive oxygen species generation and implicates Bax translocation, mitochondrial cytochrome c release and caspase activation.

In the present study, we investigated the in vitro apoptotic response of leukemic cells to the cellular stress induced by homoharringtonine (HHT), a plant alkaloid with antileukemic activity which is currently being tested for treatment of acute and chronic leukemias. A comparison of leukemic cell lines with different p53 gene status revealed a considerably higher sensitivity to HHT-induced apoptosis in the cells with a wt p53, and apoptotic events in wt p53 leukemia cells (MOLT-3 cell line) were studied in more detail. To this end, we examined components of apoptotic cascades including Bax expression and its intracellular localization, changes of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, cytochrome c release from mitochondria and activation of caspases. Bax protein levels did not increase despite an up-regulation of bax at mRNA level. However, Bax translocation from cytosol towards mitochondria was observed. In addition, we observed a release of cytochrome c from the mitochondria, and the localization changes of both Bax and cytochrome c were found already at the early, annexin V-negative stage of HHT-induced apoptosis. HHT-treated MOLT-3 cells revealed loss of MMP as well as activation of caspases demonstrated by DEVD-, IETD- and LEHD-tetrapeptide cleavage activity in the cell lysates. ROS levels only slightly increased in HHT-treated cells and antioxidants did not prevent apoptosis and MMP changes. Therefore, wt p53 leukemic cells respond to HHT-specific cellular stress by induction of ROS-independent apoptotic pathway characterized by translocation of Bax, mitochondrial cytochrome c release and activation of caspases.

Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1.

Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies, occurring in approximately 5–10% of acute lymphoblastic leukemia (ALL) and 5% of acute myeloblastic leukemia (AML). In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. The human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, as detected by the monoclonal antibody (moab) 7.1, was shown to be expressed on leukemic cells with MLL rearrangements of children with acute leukemia. We further investigated the reactivity of the moab 7.1 on 533 cell samples of adults (nu2009=u2009215) and children (nu2009=u2009318) with acute leukemias (271 AML, 217 B-lineage ALL, 37 T-lineage ALL, eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias) by flow cytometry. In AML, 38 samples were positive for moab 7.1 (‘20%-cut-off-level’). These moab 7.1-positive AML cases revealed a myelomonocytic-differentiated immunophenotype with coexpression of the NK cell marker CD56 in 33 of 38 cases. Two of eight cell samples of the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia entity reacted with moab 7.1. In ALL, 35 samples mostly of the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL) were positive for moab 7.1. 58 (81%) of 72 samples with MLL rearrangements were positive for moab 7.1 including 28/31 with a t(4;11), 16/17 with a t(9;11), 3/5 with a t(11;19), and 2/6 with a del(11)(q23). All moab 7.1-positive ALL (nu2009=u200934) and childhood AML (nu2009=u200917) cases revealed MLL rearrangements as detected by Southern blot analysis and RT-PCR. However, 11 adults with AML, and one adult with moab 7.1-positive CD7+ CD56+myeloid/natural killer cell precursor acute leukemia were negative for MLL rearrangements as proved by Southern blot analysis. We conclude that moab 7.1 is a sensitive but not entirely specific marker for the identification of 11q23-associated AML and ALL by flow cytometry in children and adults.

Constitutive expression levels of CD95 and Bcl-2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia

CD95 (Fas/APO‐1) expression and function and Bcl‐2 expression, as well as spontaneous apoptosis in vitro, have been shown to be predictive markers for the in vivo response to chemotherapy in acute myeloid leukaemia (AML). To determine the clinical significance of apoptosis‐regulating factors in acute lymphoblastic leukaemia (ALL), we investigated cell samples of children with ALL who had been included in the German ALL Berlin–Frankfurt–Münster (BFM) study using flow cytometry for constitutive expression levels of CD95 (nu2003=u2003110) and Bcl‐2 (nu2003=u2003110). Furthermore, we determined the extent of spontaneous apoptosis in vitro (nu2003=u2003102) and susceptibility to anti‐CD95‐induced apoptosis (CD95‐sensitivity) (nu2003=u200397). We correlated these findings with the functional activity of the multidrug resistance (MDR)‐associated P‐glycoprotein (P‐gp), as detected by the rhodamine123 efflux test, immunophenotype, cytogenetics and clinical data of the patients examined. Good responders to initial prednisone therapy (‘prednisone response’) revealed significantly higher Bcl‐2 expression levels [5·4u2003±u20033·4 relative fluorescence intensity (RFI), nu2003=u200368] than poor responders (3·7u2003±u20032·6 RFI, nu2003=u200342; Pu2003=u20030·002). There was no significant correlation between the other investigated parameters and prednisone response. Moreover, neither the CD95 and Bcl‐2 expression levels nor the extent of spontaneous apoptosis in vitro, CD95 sensitivity or P‐gp function were correlated with the response to induction chemotherapy or relapse rate, either for B‐cell precursor ALL or T‐cell ALL. No consistent pattern of change in CD95 (nu2003=u200310) and Bcl‐2 expression (nu2003=u20039) was noted in cases studied at both initial diagnosis and relapse. In conclusion, our findings underline the different cell biological features of primary AML and ALL cells.

Differential CD95 expression and function in T and B lineage acute lymphoblastic leukemia cells

CD95 (Fas/APO-1) is a cell surface receptor able to trigger apoptosis in a variety of cell types. The expression and function of the CD95 antigen on leukemic blasts from 42 patients with B lineage and 53 patients with T lineage acute lymphoblastic leukemia (ALL) were investigated using immunofluorescence staining and apoptosis assays. The CD95 surface antigen was expressed in most ALL cases, with the T lineage ALL usually showing a higher intensity of surface CD95 expression as compared with the B lineage ALL cells (relative fluorescence intensity, RFI: 4.8u2009±u20090.47 vs 2.2u2009±u20090.23, respectively, Pu2009<u20090.01). Functional studies disclosed that upon oligomerization by anti-CD95 monoclonal antibodies the CD95 protein was either not able to initiate apoptosis of leukemic cells (75% of cases) or induced low rates of apoptosis (20% of cases). Only in 5% of cases did the apoptosis rate exceed the 20% level of the CD95-specific apoptosis. Most of the CD95-sensitive cases were found among T lineage ALLs (38% of T lineage vs 10% of B lineage ALLs). Overall, the extent of CD95-induced apoptosis did not correlate with the expression level of CD95. Similarly, no significant correlation between expression level and functionality of CD95 in human leukemia cell lines of B and T cell origin could be observed. Bcl-2 protein has been associated with prolonged cell survival and has been shown to block partially CD95-mediated apoptosis, but for ALL cells no correlation between bcl-2 expression and spontaneous or CD95-mediated apoptosis could be found. The results obtained in this study indicate that, despite constitutive expression of CD95, the ALL cells are mainly resistant to CD95-triggering. More detailed investigations of the molecular mechanisms involved in the intracellular apoptotic signal transduction, such as interactions of the bcl-2 and the other members of the bcl-2 family, and functionality of the interleukin-1β converting enzyme (ICE) like-proteases, may give new insights into key events responsible for the resistance or sensitivity to the induction of apoptosis in acute leukemia.

In Vitro Sensitivity to Chemotherapy-Induced Apoptosis in CD2+ve and CD2-ve Childhood T-All

T-lineage acute lymphoblastic leukemia (T- ALL) account for approximately 15% of all childhood acute leukemias and has been reported to have a worse prognosis than B- lineage ALL (Reiter et al., 1994; Uckun et al., 1998). However, the reasons for the increased drug resistance of T-ALLcells are not clear yet (Uckun et al., 1998).

Involvement of BCL-2 and Bax in the IL-7-lnduced Inhibition of Spontaneous Apoptosis in Childhood T-ALL

Induction of apoptosis by cytokine deprivation and its inhibition by cytokines in growth factor-dependent cell lines has been demonstrated to be under the control of Bcl-2-related proteins. Recently, we have demonstrated that leukemic cells of patients with newly diagnosed T-ALL underwent spontaneous apoptosis upon culturing in vitroand that this kind of apoptosis could be effectively inhibited by IL-7. In the present study, we addressed the role of Bcl-2 and Bax proteins, the two major members of the Bcl-2 family, in the regulation of spontaneous and IL-7-modulated apoptosis in T-ALL. To this end, we investigated leukemic blasts from childhood T-ALL patients as to expression levels of Bcl-2 and Bax, which were quantified by flow cytometry in units of molecules of equivalent soluble fluorochrome (MESF) before and after the treatment with IL-7 (100 U/ml, 24h, 37°C), and their relative changes were correlated with the extent of apoptosis in leukemic cells in vitro.We found that expression changes of Bax, but not Bcl-2, positively correlated with the extent of spontaneous apoptosis (Spearman correlation: p=0.022 and p=0.991, respectively; 20 patients). By contrast, changes of Bax expression levels did not correlate with the inhibition of apoptosis by IL-7 (p=0.47, 30 patients). However, inhibition of spontaneous apoptosis by IL-7 was associated with upregulation of Bcl-2 and, even stronger, with Bcl-2/Bax ratios (linear regression: r=0.51, p=0.004 and r=0.59, p=0.0006, respectively, 30 patients). Therefore, our data suggest a differential involvement of Bcl-2 and Bax in the induction of spontaneous apoptosis and its regulation by IL-7 in T-ALL.