Bcr-Abl reduces endoplasmic reticulum releasable calcium levels by a Bcl-2–independent mechanism and inhibits calcium-dependent apoptotic signaling

Abstract

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis and that this plays a significant role in the achievement of drug resistance. We have established cell lines expressing low and high levels of Bcr-Abl to study the molecular mechanisms involved in disease progression and drug resistance. It is now known that the endoplasmic reticulum (ER) can play a major role in the regulation of apoptosis. We therefore investigated whether Bcr-Abl expression modulates ER homeostasis and interferes with ER-mediated apoptotic pathways to promote survival. Bcr-Abl-expressing cells exhibit a decreased amount of free releasable calcium in the ER as well as a weaker capacitative calcium entry response, relative to parental cells. This effect is independent of Bcl-2, which is a known modulator of ER calcium homeostasis. The reduction in ER releasable calcium results in inhibition of the ER/mitochondriacoupling process and mitochondrial calcium uptake. This study demonstrates a novel downstream consequence of BcrAbl signaling. The ability to negate calcium-dependent apoptotic signaling is likely to be a major prosurvival mechanism in Bcr IntroductionChronic myeloid leukemia (CML) is a clonal disorder of the hematopoietic system resulting in the excessive accumulation of immature and mature myeloid cells. The hallmark of CML is the presence of the Philadelphia (Ph) chromosome, which is present in more than 95% of cases and harbors the chimeric gene BCR-ABL. The resultant tyrosine kinase, Bcr-Abl, constitutively activates several signal transduction pathways 1,2 and is believed to be the critical determinant in the pathogenesis of CML.CML is a biphasic disease, starting as an indolent chronic phase followed by a much more aggressive and drug-resistant phase called blast crisis. A specific inhibitor of Bcr-Abl tyrosine kinase activity, imatinib (Gleevec, Glivec) has recently been introduced for the treatment of CML. 3,4 Impressive responses have been seen with patients in chronic phase; however, resistance is common in advanced CML. Reported resistance mechanisms are primarily mutations and amplification of the BCR-ABL gene. [5][6][7][8] A number of groups have suggested that the reason blast crisis (BC) CML is multidrug resistant is due to the antiapoptotic properties of the Bcr-Abl oncoprotein. It has already been shown that BCR-ABL can confer drug resistance when it is transfected into other leukemic cell lines. 9 Several clinical and molecular studies have suggested that the expression levels of Bcr-Abl may play a major role in resistance to apoptosis. 10-13 Down-regulation of Bcr-Abl transcript has been shown to increase drug sensitivity in cells derived from CML. 14 Expression of Bcr-Abl at different levels in hematopoietic progenitor cells found that all cells became growth factor independent, but only the high Bcr-Abl-expressing cells were resistant...

Release of intracellular calcium primes chronic myeloid leukaemia cells for tyrosine kinase inhibitor-induced apoptosis

“…BCR-ABL expression modulates ER calcium homeostasis, leading to interference with ER-mediated apoptotic pathways and thereby promoting cell survival, and this effect on ER-releasable calcium is greater than that on CCE. 16 Decreasing ER calcium concentration may increase HeLa cell survival in response to oxidant stress induced by ceramide. 29 Double knockout Bax and Bak (pro-apoptotic proteins) mouse fibroblasts displayed a reduced resting ER calcium concentration compared with wild-type cells, and were resistant to induction of apoptosis.…”

“…Effects of amantadine and prazosin on intracellular calcium changes BCR-ABL expression confers a deficiency in a calciumdependent apoptotic pathway, 16 while amantadine modulates intracellular calcium mobilisation 20 and improves cell membrane permeability. 21 We therefore investigated whether amantadine and prazosin pre-treatment was acting via an effect on intracellular calcium.…”

“…16 Treatment of BCR-ABL-positive cells with imatinib restored ER calcium levels and induced apoptosis. 16 Furthermore, imatinib has also been reported to induce cell death in a gastrointestinal stromal tumour cell line 17 and in murine myeloid progenitors transduced with BCR-ABL through an ER-mediated stress response. 18 In the course of investigations of how one TKI modifies the transport of another, we observed by chance that amantadine and prazosin primed CML cells for TKI-mediated cytotoxicity.…”

“…[11][12][13][14][15] BCR-ABL expression has been reported to modulate calcium homeostasis in the ER, and interfere with ER-mediated apoptotic pathways to promote survival of CML cells. 16 Changes in either the prevailing ER calcium levels and/or resultant calcium influx into the ER (known as capacitative calcium entry (CCE)) may compromise calcium-dependent apoptotic mechanisms in BCR-ABL-expressing cells. 16 Treatment of BCR-ABL-positive cells with imatinib restored ER calcium levels and induced apoptosis.…”

“…16 Changes in either the prevailing ER calcium levels and/or resultant calcium influx into the ER (known as capacitative calcium entry (CCE)) may compromise calcium-dependent apoptotic mechanisms in BCR-ABL-expressing cells. 16 Treatment of BCR-ABL-positive cells with imatinib restored ER calcium levels and induced apoptosis. 16 Furthermore, imatinib has also been reported to induce cell death in a gastrointestinal stromal tumour cell line 17 and in murine myeloid progenitors transduced with BCR-ABL through an ER-mediated stress response.…”