Attenuated AMPA Receptor Expression Allows Glioblastoma Cell Survival in Glutamate-Rich Environment

Background: Glioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na+ and Ca2+-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment. Methods and Findings: In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited. Conclusions: Together with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumor

Upregulation of SIRPα upon differentiation of t(15;17) NB4 cells and induction of cell death following its triggering.

<p>(A) NB4 cells were exposed to 1 µM ATRA and granulocytic differentiation of the cells was examined by cell surface expression of the common myeloid marker, CD11b. (B) SIRPα protein expression, determined by western blotting, is upregulated in ATRA-incubated NB4 cells. β-actin is used as a loading control. (C) Flow cytometric analysis of chSIRPα surface expression is determined by using ED9 mAb in transduced NB4 empty vector and chSIRPα expressing cells. (D) 24 hrs following ED9 (10 µg/ml) incubation, the percentage of cell death in chSIRPα and EV transduced NB4 cells was quantified by APC-Annexin V and PE-7AAD FACS staining. (E) Percentage of apoptosis after exposure to 1 µM ATRA is shown in combination with 10 µg/ml of ED9.</p

SIRPα-derived signal synergizes with different antileukemic drugs.

<p>Inhibition of cell growth is depicted by combination of ED9 mAb (10 µg/ml) with (A) Ara-C and DNR in NB4 cells expressing chSIRPα (B) Ara-C, DNR, VP16, DAC and imatinib in Kasumi-1 cells expressing chSIRPα. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052143#s3" target="_blank">Results</a> are based on means of 3 experiments and are calculated using Calcusyn.</p

SIRPα upregulation in t(8;21) Kasumi-1 cells following treatment with inhibitors of epigenetic gene silencing.

<p>Kasumi-1 cells were incubated with 1 µM Decitabine, 0.5 mM valproic acid, 1 mM Butyrate and 300 nM Trichostatin. Endogenous SIRPα protein level, determined by Western blotting was upregulated at indicated time points. β-actin staining was used as a loading control.</p

SIRPα protein expression in AML cell lines and patients.

<p>Western blot analysis was performed in (A) cell lines and (B) 20 pediatric AML patient samples. β-actin staining was used as loading control. (C) SIRPα expression is quantified relative to β-actin expression.</p

Ligation of chSIRPα induces caspase 3-independent PCD in Kasumi-1 cells.

<p>(A) Flow cytometric analysis of SIRPα expression was performed by using ED9 mAb in stable Kasumi-1 cells expressing chSIRPα and EV. (B) Kasumi-1 chSIRPα and EV cells were incubated with 10 µg/ml ED9 mAb and the percentage of cell death was determined after 24 hrs. Annexin V and 7-AAD FACS staining defined that ligation of chSIRPα resulted in increased cell death in chSIRPα Kasumi-1 cells compared to EV control cells. Data are means ± SD calculated from 3 independent experiments using triplicate samples (*: significant difference <i>p</i><0.05). (C) Kasumi-1 cells expressing chSIRPα or EV were treated with 10 µg/ml ED9 for mentioned time points. Caspase 3 staining shows no cleavage of the p32 subunit. As a positive control for caspase 3 cleavage, human neutrophils (PMN) were incubated at room temperature for 0 and 24 hours.</p

Engagement of SIRP alpha Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

<p>Background: Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRP alpha) on macrophages. Although AML cells express SIRP alpha, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRP alpha in acute myeloid leukemia.</p><p>Design and Methods: We analyzed the expression of SIRP alpha, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRP alpha on two low SIRP alpha expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRP alpha expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.</p><p>Results: By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRP alpha is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0-M3. Interestingly, AML patients with high SIRP alpha expression had a poor prognosis. Our results also showed that SIRP alpha is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRP alpha with an agonistic antibody in the cells stably expressing chimeric SIRP alpha, led to inhibition of growth and induction of programmed cell death. Finally, the SIRP alpha-derived signaling synergized with the activity of established antileukemic drugs.</p><p>Conclusions: Our data indicate that triggering of SIRP alpha has antileukemic effect and may function as a potential therapeutic target in AML.</p>