Genomic Mechanisms of p210BCR-ABL Signaling

Abstract

Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210 BCR-ABL oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210 BCR-ABL versus those expressing an empty vector using cDNA macro-and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210 BCR-ABL ), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210 BCR-ABL in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210 BCR-ABL responsive element to a high affinity 5 (A/T)GATA(A/G)-3 "GATA" response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210 BCR-ABL -and p185 BCR-ABL -mediated trans-activation to an inert promoter. Short interfering RNA mediated "knockdown" of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein.We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.

Inhibition of the Mitotic Kinesin Eg5 Up-regulates Hsp70 through the Phosphatidylinositol 3-Kinase/Akt Pathway in Multiple Myeloma Cells

“…To examine whether dimethylenastron also regulates Hsp70 transcriptionally, we performed a luciferase reporter assay using pHsp70(Ϫ259), a luciferase reporter plasmid driven by Hsp70 promoter (28). Consistent with the results obtained from Western blot analysis, dimethylenastron treatment increased the pHsp70(Ϫ259) luciferase activity (Fig.…”

“…To further characterize the transcriptional regulation of Hsp70 by dimethylenastron, we transfected cells with a series of luciferase reporter plasmids driven by different sizes of Hsp70 promoters, including pHsp70(Ϫ259), pHsp70(Ϫ200), pHsp70(Ϫ163), pHsp70(Ϫ123), and pHsp70(Ϫ82), which differed in size because of 5Ј deletions (28). Dimethylenastron induced comparable luciferase activities in pHsp70(Ϫ259), pHsp70(Ϫ200), and pHsp70(Ϫ163) but significantly reduced activities in pHsp70(Ϫ123) and pHsp70(Ϫ82) (Fig.…”

“…Luciferase Reporter Assay-Cells were transfected with pRL-TK Renilla luciferase reporter plasmid (Promega) and Hsp70 promoter- driven firefly luciferase reporter plasmids (28). Luciferase activities were then determined by using the dual luciferase reporter assay system according to the manufacturer's protocol (Promega).…”

The heat shock proteins as targets for radiosensitization and chemosensitization in cancer

“…4,9,60,61 One study investigated serum Hsp70 compared with serum PSA in detecting early stage prostate cancer, and demonstrated a significant correlation between serum protein levels and disease. 62 Further, patients with CML expressing the chimeric oncogene Bcr-Abl were also found to harbor high levels of Hsp70, 63 suggesting a role for the chaperone in the stability of the protein in vivo. Such a finding was particularly striking in patients with imatinib-resistant CML.…”

Heat shock protein-70 neutralizes apoptosis inducing factor in Bcr/Abl expressing cells

“…Additionally, induction of HSP-70 was not accompanied with HSF-1 amount and phosphorylated status, indicating that the Bcr/Abl-mediating HSP-70 induction may be HSF-1 independent. Recently, a study demonstrates that the fusion protein induces HSP-70 through GATA-1, a trans-factor that binds GATA response element upstream HSP-70 promoter in Bcr/ Abl expressing HL-60 cells [45]. GATA-1 plays a central role in the differentiation of erythrocytes and megakaryocytes and is required for the expression of globin, heme biosynthesis, and red blood cell transcription factor genes [46].…”