Identification of genes involved in Ca2+ ionophore A23187-mediated apoptosis and demonstration of a high susceptibility for transcriptional repression of cell cycle genes in B lymphoblasts from a patient with Scott syndrome

Abstract

Background: In contrast to other agents able to induce apoptosis of cultured cells, Ca2+ ionophore A23187 was shown to elicit direct activation of intracellular signal(s). The phenotype of the cells derived from patients having the hemorrhagic disease Scott syndrome, is associated with an abnormally high proportion of apoptotic cells, both in basal culture medium and upon addition of low ionophore concentrations in long-term cultures. These features are presumably related to the mutation also responsible for the defective procoagulant plasma membrane remodeling. We analyzed the specific transcriptional re-programming induced by A23187 to get insights into the effect of this agent on gene expression and a defective gene regulation in Scott cells.

Results: The changes in gene expression upon 48 hours treatment with 200 nM A23187 were measured in Scott B lymphoblasts compared to B lymphoblasts derived from the patient's daughter or unrelated individuals using Affymetrix microarrays. In a similar manner in all of the B cell lines, results showed up-regulation of 55 genes, out of 12,000 represented sequences, involved in various pathways of the cell metabolism. In contrast, a group of 54 down-regulated genes, coding for histones and proteins involved in the cell cycle progression, was more significantly repressed in Scott B lymphoblasts than in the other cell lines. These data correlated with the alterations of the cell cycle phases in treated cells and suggested that the potent effect of A23187 in Scott B lymphoblasts may be the consequence of the underlying molecular defect.

Conclusion: The data illustrate that the ionophore A23187 exerts its pro-apoptotic effect by promoting a complex pattern of genetic changes. These results also suggest that a subset of genes participating in various steps of the cell cycle progress can be transcriptionally regulated in a coordinated fashion. Furthermore, this research brings a new insight into the defect in cultured Scott B lymphoblasts, leading to hypothesize that a mutated gene plays a role not only in membrane remodeling but also in signal transduction pathway(s) leading to altered transcriptional regulation of cell cycle genes.

Figure 1

Cluster analysis to compare the fold changes in gene expression in cells treated by A23187. Cluster analysis allowed to directly compare the fold changes in gene expression for each one the three cell lines respectively (C: control B lymphoblasts; D: daughter's B lymphoblasts; S: Scott B lymphoblasts). Ratios determining the fold changes in gene expression due to treatment for 48 hours with 200 nM A23187 were examined for each one of the three cell types. The hierachical clustering of the selected genes (fold change of gene expression >2 with a P-value of <0.02 in at least one of the three cell types, see additional file 1) was performed. Without changing their respective hierarchical order, the genes were then grouped by functional category. Differential gene expression in A23187-treated versus non-treated cells is color coded as indicated at the bottom of the figure (fold-changes < 0.5 [see Additional file 1] correspond here to <-1/0.5, i.e. <-2). (A) Cluster analysis of up-regulated genes. (B) Cluster analysis of down-regulated genes. For selected genes represented on the U95Av2 GeneChips by more than one Probe Set ID [see Additional file 1], data corresponding to a single Probe Set ID were used for the cluster analysis and the gene lists in Tables 2 and 3.

Figure 2

Quantitative RT-PCR validation for a subset of genes differentially expressed by treatment with A23187. RTs were performed with new RNA prepared from the treated and untreated cell lysates. For each PCR experiment, RT samples were analyzed at least in triplicate for the expression of a gene in parallel with GAPDH and 18S rRNA as described in Methods. Results are means ± SD from two independent PCR experiments with different RTs (each analyzed at least in triplicate) for the up-regulated genes and three independent PCR experiments for the down-regulated genes. The fold changes in expression level, i.e. 2^-ΔΔCT for the up-regulated genes calculated as described in Methods and -1/2^-ΔΔCT for the down-regulated genes (to compare with the values given in Table 3), are represented on a logarithmic scale. Some of the gene symbols listed in Table 2 and 3 have been recently renamed by Affymetrix [54] and the latest symbols were used for ordering the Assays-on-Demand. WARS: IFN-inducible gamma 2, Ac. N° X59892; ASS: argininosuccinate synthetase, Ac. N° X01630; SIAH2, Ac. N° U76248; CDC20: p55CDC, Ac. N° U05340; CCNB1: cyclin B, Ac. N° M25753; AURKB: protein kinase STK 12, Ac. N° AF004022; UBE2C: cyclin-selective ubiquitin carrier, Ac. N° U73379; H2H2AA: H2A.2, Ac. N° L19779; H1H2BM: H2B/e, Ac. N° Z83738.

Figure 3

Effect of A23187 on B lymphoblasts cell cycle. The B lymphoblasts cultured in the absence or presence of 200 nM A23187 for 48 hours were stained with PI and analyzed for the proportion of cells in the different phases of the cycle by flow cytometry as described in Methods. Two independent experiments were performed with each one of five cell lines (two Scott B lymphoblasts independently immortalized from the patient's lymphocytes, one daughter's and two control cell lines from unrelated individuals). Data are means ± SD from the four independent experiments for Scott and control B lymphoblasts respectively and the two experiments for daughter's cells. For a given phase of the cell cycle and a cell type, P reflects the statistical differences between the percent of cells in the presence versus absence of A23187. * P = 0.0038, # P = 0.0016, † P = 0.0344, ★ P = 0.004, ◆ P = 0.0134.