p53-Dependent Transcriptional Responses to Interleukin-3 Signaling

p53 is critical in the normal response to a variety of cellular stresses including DNA damage and loss of p53 function is a common feature of many cancers. In hematological malignancies, p53 deletion is less common than in solid malignancies but is associated with poor prognosis and resistance to chemotherapy. Compared to their wild-type (WT) counterparts, hematopoietic progenitor cells lacking p53 have a greater propensity to survive cytokine loss, in part, due to the failure to transcribe Puma, a proapoptotic Bcl-2 family member. Using expression arrays, we have further characterized the differences that distinguish p53−/− cells from WT myeloid cells in the presence of Interleukin-3 (IL-3) to determine if such differences contribute to the increased clonogenicity and survival responses observed in p53−/− cells. We show that p53−/− cells have a deregulated intracellular signaling environment and display a more rapid and sustained response to IL-3. This was accompanied by an increase in active ERK1/2 and a dependence on an intact MAP kinase signaling pathway. Contrastingly, we find that p53−/− cells are independent on AKT for their survival. Thus, loss of p53 in myeloid cells results in an altered transcriptional and kinase signaling environment that favors enhanced cytokine signaling

Analysis of the IL-3 signaling pathway in WT and <i>p53^−/−</i> FDM cells.

<p>(A) Independently generated WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were cultured in the indicated concentrations of IL-3 for 72 hours. Viability was determined using Propidium iodide (PI) exclusion detected by flow cytometry. Results show the mean +/− SEM of 3 independent experiments. (B) WT and <i>p53^−/−</i> independent FDM cell lines (n = number of cell lines) were cultured in the indicated concentrations of IL-3 doses for 72 hours and then plated in soft agar with abundant IL-3. The number of colonies was counted after 14 days and the clonogenicity (relative to the number of colonies generated in 500 pg/ml IL-3) calculated. Results show the mean +/− SEM of 2 independent experiments. (C) Lysates were generated from WT pr <i>p53^−/−</i> FDM cells following stimulation with the indicated concentrations of IL-3 after 16 hours of IL-3 deprivation. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific to the indicated proteins. (D) Independent WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were treated for 24 h with an AKT inhibitor (AKTi) in the presence or absence of IL-3. Viability was determined by flow cytometric analysis of PI exclusion. Results show the mean +/− SEM of 2 independent experiments. (E) Independent WT and <i>p53^−/−</i> FDM cell lines (n = number of cell lines) were treated for 24 h with a MEK inhibitor (MEKi) in the presence or absence of IL-3. Viability was determined by flow cytometric analysis of PI exclusion. Results show the mean +/− SEM of 4 independent experiments.</p

Pathway analysis of WT and <i>p53^−/−</i> samples cultured in the presence of cytokine.

<p>(A) List of activated and inactivated pathways identified by a Signaling Pathway Impact Analysis (SPIA) of array results from WT and <i>p53^−/−</i> samples. The ID is the KEGG ID, pSize indicates the number of genes in the KEGG pathway, NDE is the number of differentially expressed genes found within the pathway and pGFdr is the False Discovery pathways (FDR<0.1). Significant pathways are shown. (B) The differentially expressed genes that account for the significant SPIA pathways (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031428#pone-0031428-g002" target="_blank">Figure 2A</a>) are depicted by the heatmap. To the right of the heatmap, the dots indicate the pathways to which each gene contributes and whether a gene is represented by several of the pathways.</p

Proportion of expressed probes for each array.

<p>Proportion of expressed probes for each array.</p

Differential gene expression in WT and <i>p53^−/−</i> FDM samples.

<p>(A) RNA from three independent WT and <i>p53^−/−</i> FDM cell lines were analyzed with the use of the 6-chip Illumina expression array. The heatmap depicts the expression of the top 30 differentially expressed genes according to the adjusted P value in WT and <i>p53^−/−</i> samples. (B) Differentially expressed genes highly expressed in <i>p53^−/−</i> compared to WT samples with a logFC change of greater than 2 are shown. (C) Differentially expressed genes highly expressed in WT compared to <i>p53^−/−</i> samples with a logFC change of greater than 2 are shown. Asterisks show the p53-dependent gene CDKN1A (p21).</p

Gene set enrichment analysis of WT and <i>p53^−/−</i> expression array data.

<p>(A) Gene Set Enrichment Analysis (GSEA) of the WT and <i>p53^−/−</i> gene lists at the various time points. GSEA was conducted using all genes that were considered expressed in the array, based on their detection p-value. A p-value of less than 0.05 was considered significant. (B) (C) Each diamond represents an individual probe for significantly differentially expressed kinases (B) or transcription factors (C) in WT and <i>p53^−/−</i> FDM cells cultured in IL-3.</p

Differential pathway expression in WT samples after IL-3 loss.

<p>(A) Three independent WT and <i>p53^−/−</i> FDM cell lines were culture with or without IL-3 for 6 h. RNA was extracted and expression array was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031428#pone-0031428-g001" target="_blank">Figure 1A</a>. The heat map shows significant changes in expression after IL-3 deprivation in WT cells and dots indicate the various SPIA pathways these represent. 31 differentially expressed genes were active in these pathways, with 24 being highly expressed at time zero and 12 highly expressed at 6 h IL-3 withdrawal. Significant pathways are shown (FDR<0.1). (B) Comparison of SPIA of array results from WT and <i>p53^−/−</i> samples after IL-3 withdrawal. The ID is the KEGG ID, pSize indicates the number of genes in the KEGG pathway, NDE is the number of differentially expressed genes found within the pathway and pGFdr is the False Discovery pathways (FDR<0.1). Significant pathways are shown.</p

Myeloid progenitor cells lacking p53 exhibit delayed up-regulation of Puma and prolonged survival after cytokine deprivation

Loss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)–dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53−/−, and FoxO3a−/− FDM cells and found that p53−/− but not FoxO3a−/− cells were protected against IL-3 withdrawal. Loss of p21cip/waf, which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53−/− hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53−/− cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis