Abstract
The paradox of peripheral cytopenias despite of normo/hypercellular marrow in myelodysplastic syndrome (MDS) has been ascribed to excessive intramedullary hematopoietic cell apoptosis. Several apoptosis-inducing systems, including Fas/Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, are upregulated in MDS. FLIP (FLICE (FAS-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) was identified as a FAS and TRAIL signal inhibitor. The largest variant FLIPLong (FLIPL) was originally characterized as a molecule with inhibitory activity on caspase-8. The short splice form termed FLIPShort (FLIPS) has also been characterized as a potent (TRAIL-induced) apoptosis inhibitor. However, whereas FLIPL and FLIPS have been described as death receptor pathway inhibitors, recent data suggest that physiologically, FLIPL may have caspase-8-activating properties. This study aims to characterize the expression of FLIPL and FLIPS based on mRNA, by Real-time quantitative PCR, in marrow cells from MDS patients and to correlate the expression with French-American-British (FAB) and World Health Organization (WHO) classification. For each sample, results were first calculated as a ratio of the total transcript number of FLIPL or FLIPS and the total transcript number of the endogenous reference gene (β-actin) to obtain a normalized target value. Transcript ratios of each sample were normalized against the respective ratio of a pool of 6 normal bone marrow donors (NBM), and the ratio between the two was used as measure for the relative FLIPL or FLIPS level. We hypothesized that FLIPL and FLIPS expression differed between low and high risk of MDS. Marrow aspirates were obtained from 6 NBM and 16 patients with MDS out of treatment (7 males, 9 females; 23–78 (median 64) yo). The National Ethical Committee Board approved this study, informed-written consent was obtained from all patients and donors. According to FAB classification, patients were distributed as: 10 RA, 2 RARS and 4 RAEB. According to WHO classification: 10 RCMD, 2 RCMD-RS, 3 RAEB-1 and 1 RAEB-2. FLIPS mRNA expression were significantly higher in high risk DS according to FAB and WHO classification; RA/RARS compared with AREB (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03). However, FLIPL mRNA expression also tended to be higher in high risk MDS according to FAB and WHO classification, though not significantly different: RA/RARS compared with AREB (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46). Lower FLIPS level in low risk MDS marrows, in addition to the well described upregulation of extracellular proapoptotic signals, would explain the increased susceptibility of hematopoietic cells in low risk MDS marrow to death-inducing stimuli. The fact that FLIPL expression did not differ according to FAB and WHO classification could be related to the hypothesis that FLIPL may have caspase-8-activating properties rather than anti-apoptotic activity. Differential regulation of FLIPL and FLIPS according to risk groups in MDS patients might result in different rates of apoptosis. Further studies are needed to elucidate the mechanisms controlling and regulating FLIP expression in normal and malignant hemopoietic cells.
Differential effects of mitomycin C and doxorubicin on P-glycoprotein expression
