Cristina Montiel-Duarte

BCR-ABL Induces the Expression of Skp2 through the PI3K Pathway to Promote p27Kip1 Degradation and Proliferation of Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G(1) cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27(Kip1) and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias.

p38 MAPK mediates the regulation of α1(I) procollagen mRNA levels by TNF-α and TGF-β in a cell line of rat hepatic stellate cells1

The role of members of the mitogen‐activated protein kinase (MAPK) family on tumor necrosis factor α (TNF‐α)‐mediated down‐regulation of col1a1 gene was studied. TNF‐α increased extracellular‐regulated kinase and Jun‐N‐terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on α1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF‐α, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF‐α on col1a1 mRNA levels. Transforming growth factor β (TGF‐β) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF‐β. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

Abnormal methylation of the common PARK2 and PACRG promoter is associated with downregulation of gene expression in acute lymphoblastic leukemia and chronic myeloid leukemia.

The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm.

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P=0.03) and T-ALL vs B-ALL (50 vs 9%, P=0.001). Relapse rate (62 vs 44%, P=0.05) and mortality (59 vs 43%, P=0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P<0.001 y P<0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors

Despite their physiological importance, selective interactions between nuclear receptors (NRs) and their cofactors are poorly understood. Here, we describe a novel signature motif (F/YSXXLXXL/Y) in the developmental regulator BCL11A that facilitates its selective interaction with members of the NR2E/F subfamily. Two copies of this motif (named here as RID1 and RID2) permit BCL11A to bind COUP-TFs (NR2F1;NR2F2;NR2F6) and Tailless/TLX (NR2E1), whereas RID1, but not RID2, binds PNR (NR2E3). We confirmed the existence of endogenous BCL11A/TLX complexes in mouse cortex tissue. No interactions of RID1 and RID2 with 20 other ligand-binding domains from different NR subtypes were observed. We show that RID1 and RID2 are required for BCL11A-mediated repression of endogenous γ-globin gene and the regulatory non-coding transcript Bgl3, and we identify COUP-TFII binding sites within the Bgl3 locus. In addition to their importance for BCL11A function, we show that F/YSXXLXXL/Y motifs are conserved in other NR cofactors. A single FSXXLXXL motif in the NR-binding SET domain protein NSD1 facilitates its interactions with the NR2E/F subfamily. However, the NSD1 motif incorporates features of both LXXLL and FSXXLXXL motifs, giving it a distinct NR-binding pattern in contrast to other cofactors. In summary, our results provide new insights into the selectivity of NR/cofactor complex formation.

Erratum: ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia (Oncogene (2013) 32 (803) DOI: 10.1038/onc.2012.593)

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.