La estructura es el puente para asegurar que los
III. Líderes de Innovación
The aims of this Chapter were to determine the effects of imatinib on the viability of two CML cell lines, LAMA-84 and KCL-22, in order to establish their usefulness as in vitro models to test for the effects of new kinase inhibitors on the growth parameters of TKI-sensitive and -insensitive cell lines. The results in this Chapter confirm previously published results (see section 3.1) and background work in this laboratory, which has shown that LAMA-84 cells are imatinib-sensitive and that KCL-22 cells are relatively imatinib-insensitive. However, new data presented in this Chapter show that imatinib induces cell death in LAMA-84 cells by a caspase- sensitive mechanism, which is likely to be apoptosis, as this drug also induces a rapid (within a few hours) depolarisation of the mitochondrial inner membrane. Unexpectedly, the results also show that whilst imatinib did not induce cell death in KCL-22 cells, it did induce a cell cycle arrest in G1, which was not affected by the presence of the pan-caspase inhibitor.
LAMA-84 cell line was established from the blood of a patient with chronic myeloid leukaemia, in accelerated phase. It is reported to retain the patient's Philadelphia chromosome. However, recent study shows that LAMA-84 cell line has lower BCR-ABL mRNA level compared to another imatinib-resistant cell line, IRK562. Also, it has been reported that LAMA- 84 showed greater sensitivity to imatinib compared to IRK562 (Mendonca et al., 2010).
Imatinib significantly decreased LAMA-84 viability at concentrations of 5 μM and above. This finding is in line with several other studies that have used this LAMA-84 cell line as a model imatinib-sensitive CML cell line (Greene et al., 2007, Dasmahapatra et al., 2007, Mendonca et al., 2010). My results show that imatinib induced apoptosis (that was partly reversed by the pan caspase inhibitor) by increasing the number of cells that accumulated in the G0 phase (low DNA content per cell) and induced a decrease in mitochondrial membrane potential (which was also prevented by Z-VAD, data not shown). Caspase-induced activation of apoptosis is usually regulated by changes in function of Bcl-2 family member proteins,
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and so the effects of imatinib on the expression levels of several of these proteins was investigated.
For LAMA-84 cells, imatinib treatment did not result in any significant decreases in expression levels of Bcl-2 or Bcl-XL, two anti-
apoptotic members of this family. While it is possible that imatinib affected the localisation or activity of these proteins, no changes in overall levels of these proteins would indicate that they are unlikely to be affected by this drug. However, major changes in the levels of the anti-apoptotic protein, Mcl-1 were observed in LAMA-84 cells after treatment with imatinib, and these decreases were largely prevented by Z-VAD, indicating the importance of caspase activation in this process. A previous report has shown that imatinib decreased the expression of Mcl-1 in K562 cell lines (Aichberger et al., 2005) which, like LAMA-84 cells are imatinib-sensitive CML cell lines (Greene et al., 2007, Deininger et al., 1997). However, this, to my knowledge, is the first report of imatinib-induced decreases in Mcl-1 levels in LAMA-84 cells as an explanation for induction of cell death by this drug.
In contrast, imatinib had no significant effect on the viability of KCL-22 cells. However, surprisingly, it induced a partial cell cycle arrest in G1. This was insensitive to Z-VAD and hence independent of caspase activation, and has not previously been reported. It has been reported that KCL-22 cell line possesses natural resistance to imatinib (Deininger et al., 1997, Mahon et al., 2000). Therefore, this supports the idea that KCL-22 could act as a model for imatinib-insensitive CML for further studies. However, the mechanism of the resistance of KCL-22 to imatinib is still unclear. It has been reported that KCL-22 can resist imatinib without increasing of BCR-ABL expression (Mahon et al., 2000, Ohmine et al., 2003), but other mechanisms might involve BCR-ABL-independent resistance to imatinib (Mahon et al., 2000).
However, a more recent study has shown that KCL-22 have doubled expression of both BCR-ABL mRNA and protein compared to other imatinib-resistant cell lines (Quentmeier et al., 2011). This report also
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demonstrated that there was high expression of mutated kinase activity in KCL-22 cells. This point mutation results in an amino acid change in PI3K of E545G. This suggests that this mutation might constitutively activate the PI3K pathway in KCL-22 cells (Quentmeier et al., 2011). Thus, this could partly explain the mechanism of resistance of KCL-22 cell line.
Interestingly, like mature neutrophils (Moulding et al., 2001), KCL- 22 cells did not express the anti-apoptotic protein Bcl-2. They did express Bcl-XL (unlike mature neutrophils) and Mcl-1 (like mature neutrophils), but
imatinib did not induce any changes in expression levels of these latter two proteins, in line with its inability to induce apoptosis.
Another important finding from this study was the usefulness of measuring mitochondrial membrane polarisation using the fluorescence indicator JC-1. This dye is cell permeable and “stacks” in the inner membrane of polarised mitochondria (Reers et al., 1995). These JC-1 aggregates in polarised membranes emit red fluorescence (590 nm) when excited at 499 nm. However, upon membrane depolarisation, these aggregates dissociate to the monomeric form of JC-1, which emits green fluorescence (530 nm). This red:green shift can be detected by flow cytometry. An advantage of using this technique to measure apoptosis, is that changes in mitochondrial inner membrane potential are amongst the first cellular changes to be detected during activation of apoptosis. Interestingly, changes in JC-1 fluorescence in LAMA-84 cells were detected within 2-4 h of incubation with imatinib, making it suitable for rapid screening new molecules with the potential to induce apoptosis.
Taken together, the results obtained in this Chapter confirm that these two cell lines can be used as experimental tools to study imatinib- sensitive and imatinib-insensitive CML cells.
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