ALBERT

Description: Abstract 2889 GSK-3β is a multifunctional kinase that plays a role in several signaling pathways. Due to the contradictory roles of GSK-3β as a mediator of both cell survival and apoptosis, we have examined the role of GSK-3β for proliferation and apoptosis in leukemic cell lines KG1a, K562 and CMK. GSK-3β was selectively inhibited by the small-molecule SB-415286. Treatment of leukemia cells with SB-415286 (40 μM) for 72 hr approximately halved cell growth in all three cell lines. SB-415286 also showed a concentration-dependent stabilization of intracellular β-catenin: In KG1a cells the mean fluorescence intensity (MFI) [± 95% CI] was 3.1 [± 1.7] in untreated cells vs. 423 [± 24] in treated cell. The figures for the K562 and CMK cell lines were: 2.8 [± 1.6] vs. 353.2 [± 11.1], and 6.8 [± 4.0] vs. 320.2 [± 23.7], respectively. Cell cycle analysis was carried out to examine if the growth inhibition was caused by arrest in cell cycle and/or induction of apoptosis. We found that SB-415286 caused cell cycle arrest in the G2/M phase and accumulation of events corresponding to the subG1 phase, indicative of DNA fragmentation. The subG1 population was 45%, 34% and 17% in KG1a, K562 and CMK cells, respectively. To confirm that the increase of the subG1 fraction represented an apoptotic effect of the GSK-3β inhibition, we analyzed phosphatidylserine (PS) externalization and plasma membrane integrity. We found that SB-415286 caused a considerable increase of the proportion of early apoptotic cells, i.e. cells that were annexin V-positive and 7-AAD-negative: Mean [± 95% CI] in KG1a cells increased from 6.2% [± 1.2%] in untreated cells to 38% [± 3.1%] in treated cells. The figures for the K562 and CMK cell lines were: 3.0% [± 1.2%] vs. 29% [± 3.3%], and 3.9% [± 1.0%] vs. 16.0% [± 1.1%], respectively. Apoptosis signaling can be initiated by extracellular (death receptor) and/or intracellular (mitochondrial) signals. Flow cytometric analysis of cells stained by a dual-fluorescent mitochondrial dye JC-1 showed that 5–11% of untreated leukemic cells had low mitochondrial membrane potential. After 72 hr exposure to SB-415286 the mean [±95% CI] loss of the mitochondrial potential was found in 23% [± 2.0%], 33% [± 3.5%] and 42% [± 3.8%], in CMK, K562 and KG1a cells, respectively. Since drug treatment in some cell types may result in activation of both the intrinsic or extrinsic cell-death pathway in a parallel manner, we investigated if the external pathway is involved in SB-415286-induced apoptosis. For this purpose we assessed caspase-8 activation by flow cytometry. After 72 hr of treatment of CMK, K562 and KG1a cells the caspase-8 activities compared, to untreated cells, had increased 3.7-fold, 3.9-fold, and 4.4-fold, respectively. In some cell types, the extrinsic cell-death pathway leads to the cleavage of Bid (pro-apoptotic member of the Bcl-2 family) by caspase-8, generating a truncated version of the protein (tBid) which in turn activates the mitochondrial apoptotic pathway. Therefore, we determined whether depolarization of the mitochondrial membrane in the leukemic cell lines was an effect of activated caspase-8 or a direct effect of SB-415286. For this purpose Z-IETD-FMK (25 μM), a specific inhibitor of caspase-8, was applied to the cells for 2 hr. We found that inhibition of caspase-8 did not prevent SB-415286-induced apoptosis assessed by PS externalization. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Taken together, our observations suggest that inhibition of GSK-3β induces apoptosis of leukemic cells by depolarizing the mitochondria membrane. Thus, inhibition of GSK-3β could be an attractive target for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: The study's objective was to identify HPA 1a–negative women and to offer them an intervention program aimed to reduce morbidity and mortality of neonatal alloimmune thrombocytopenia (NAIT). HPA 1 typing was performed in 100 448 pregnant women. The HPA 1a–negative women were screened for anti–HPA 1a. In immunized women, delivery was performed by Cesarean section 2 to 4 weeks prior to term, with platelets from HPA 1a–negative donors reserved for immediate transfusion if petechiae were present and/or if platelet count was less than 35 × 109/L. Of the women screened, 2.1% were HPA 1a negative, and anti–HPA 1a was detected in 10.6% of these. One hundred seventy pregnancies were managed according to the intervention program, resulting in 161 HPA 1a–positive children. Of these, 55 had severe thrombocytopenia (〈 50 × 109/L), including 2 with intracranial hemorrhage (ICH). One woman with a twin pregnancy missed the follow-up and had one stillborn and one severely thrombocytopenic live child. In 15 previous prospective studies (136 814 women) there were 51 cases of severe NAIT (3 intrauterine deaths and 7 with ICH). Acknowledging the limitation of comparing with historic controls, implementation of our screening and intervention program seemed to reduce the number of cases of severe NAIT-related complications from 10 of 51 to 3 of 57.

Description: Background: Neonatal alloimmune thrombocytopenia (NAIT) is most frequently caused by antibodies against the human platelet antigen (HPA) 1a. The objective of the present study was to identify HPA 1a negative women, and to offer them an intervention program aimed to reduce morbidity and mortality of NAIT. Methods: A total of 100,448 pregnant women were HPA 1 typed. The HPA 1a negative women were screened for anti-HPA 1a, which was quantified when present. Immunized women were referred to a university hospital for clinical follow-up, including ultrasonographic examination of the fetal brain. Caesarean section was performed 2–4 weeks prior to term with platelets from HPA 1bb donors reserved for immediate transfusion if petechiae were present and/or if platelet count was 〈 35 × 109/L. Results: Of all women typed 2.1% were HPA 1a negative. Anti-HPA 1a was detected in 210 of 1,990 HPA 1a negative women. A total of 170 pregnancies in 154 HPA 1a negative women were managed according to the intervention program. These women gave birth to 161 HPA 1a positive children of whom 55 had severe thrombocytopenia (