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.
Highlights
Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210BCR-ABL oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance
We suggest that down-regulation of the GATA-heat shock protein 70 (Hsp70) pathway may be useful in the treatment of chemotherapy-resistant CML
To help identify the GATA family member interacting with the Hsp70 promoter, we examined the high density microarray data for expression of all GATA isoforms, notably GATA isoforms-1, -2, and -3, which have been implicated in hematopoiesis [43]
Summary
Cell Culture and Treatment—Human K562 erythroleukemia [16], HepG2 hepatocellular carcinoma [31], and HL-60 acute myeloid leukemia cells stably transfected with BCR-ABL (HL-60/BCR-ABL) or empty vector (HL-60/Neo) were cultured as described [23]. Comparisons of mRNA populations between control and p210BCR-ABL-expressing cells were performed with two different sets of Atlas Array membrane lots in two independent experiments. To compare differences in gene expression between arrays, background subtracted average intensity was normalized to that of housekeeping genes Those genes that showed an average 3.5-fold up-regulation or down-regulation across duplicate membranes were further considered. Oligonucleotide Microarray Data Analysis—Reproducibility of the four independent microarrays was determined by calculating the correlation coefficient for the log-transformed average difference values for the probe sets in each array [34]. Genes differentially expressed were identified by one-way ANOVA with replicates comparing the average difference values of a probe sets in HL-60/Neo versus HL-60/BCR-ABL. The average difference values for each probe set present on the array were plotted and used to calculate a correlation coefficient describing the reproducibility of the microarray data. Normalization factor used to scale the data to that of the base chip (“Experimental Procedures”)
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