A non-apoptotic function of caspase-3 in pharmacologically-induced differentiation of K562 cells

Abstract

Background and purpose: Several anticancer drugs with diverse chemical structures can induce differentiation of cancer cells. This study was undertaken to explore the potential contribution of caspase-3 to pharmacologically-induced differentiation of K562 cells.

Experimental approach: We assessed differentiation by measuring the expression of glycophorin A and haemoglobin synthesis in K562 cells treated with low concentrations of doxorubicin, hydroxyurea, cytosine arabinoside, cisplatin and haemin. Caspase-3 activation, mitochondrial membrane potential dissipation and viability were assessed by FACS. GATA-1-binding activity was evaluated by EMSA.

Key results: Treatment of K562 cells with low concentrations of the tested drugs activated caspase-3 but did not trigger detectable apoptosis. Instead, elevated levels of haemoglobin-positive and glycophorin A/caspase-3-double-positive cells were observed, suggesting involvement of caspase-3 in drug-induced differentiation. Inhibition of caspase-3 activity significantly reduced the ability of K562 cells to execute the differentiation programme. Mitochondrial membrane potential dissipation was observed, indicating involvement of the mitochondrial pathway. Binding activity of GATA-1, transcription factor responsible for differentiation and cell survival, was not diminished by increased caspase-3 activity during drug-stimulated differentiation.

Conclusions and implications: Our results could explain how anticancer drugs, with diverse structures and modes of action, can stimulate erythroid differentiation in leukaemic cells with appropriate genetic backgrounds. Our findings imply that some similarities exist between pharmacologically-induced differentiation of erythroleukaemic cells and normal erythropoiesis, both involving caspase-3 activation at high levels of anti-apoptotic protein Bcl-X(L) and chaperone protein Hsp70 (heat shock protein 70). Therefore, the functions of caspase-3, unrelated to cell death, can be extended to pharmacologically-induced differentiation of some cancer cells.

Figure 2

Activation of caspase-3 in K562 cells treated with DOX, cisPt, Ara-C, HU and haemin is not related to apoptosis. (A) Untreated cells or cells treated with 100 nM DOX, 5 µM cisPt, 250 nM Ara-C, 600 µM HU and 30 µM haemin for 4 days were stained with NucViewTM 488 caspase-3 substrate and analysed by flow cytometry (FL1 channel). PI-positive cells were excluded from the analysis. The histograms represent results from a single typical experiment. (B) Untreated cells or cells treated with tested drugs for 5 days were stained with acridine orange and ethidium bromide and examined by fluorescence microscopy. The numbers of apoptotic or necrotic cells were below 4% as assessed in 300 cells. Representative microscopic fields are shown. (C) Untreated K562 cells or cells treated with tested drugs for 4 days were stained with Annexin V and PI and analysed by flow cytometry (FL1 and FL2 channel respectively). Histograms represent results from a single typical experiment. Ara-C, cytosine arabinoside; cisPt, cisplatin; DOX, doxorubicin; FITC, fluorescein isothiocyanate; HU, hydroxyurea; PI, propidium iodide.

Figure 1

(A) Concentration-dependent effect of DOX, cisPt, Ara-C, HU and haemin on cell growth, viability and differentiation of K562 cells. Cells were cultured in the presence of various concentrations of DOX, cisPt, Ara-C, HU and haemin. After 3 days, cell growth and viability were determined. To evaluate erythroid differentiation, the percentage of haeme-containing cells was assessed by benzidine staining. Cell growth and viability were determined by Trypan blue exclusion assay. (B) Time-dependent effect of drugs on differentiation of K562 cells. Cells were cultured in the presence of selected concentrations of drugs: 100 nM DOX, 5 µM cisPt, 250 nM Ara-C, 600 µM HU and 30 µM haemin for 3, 4 and 5 days. The percentage of differentiated cells was determined by benzidine staining. (C) Time-dependent effect of drugs on viability of K562 cells. Untreated cells or cells treated for 3, 4 and 5 days with 100 nM DOX, 5 µM cisPt, 250 nM Ara-C, 600 µM HU or 30 µM haemin were stained with propidium iodide and analysed by flow cytometry. Data represent the mean ± SD of at least three independent experiments carried out in triplicate. Significant differences (P < 0.001) from the control data points were obtained for all values shown in the figure. Ara-C, cytosine arabinoside; cisPt, cisplatin; DOX, doxorubicin; HU, hydroxyurea; PI, propidium iodide.

Figure 3

Caspase-3 is involved in the pharmacologically-induced differentiation of K562 cells. (A) Untreated cells or cells treated for 3, 4 and 5 days with 100 nM DOX, 5 µM cisPt, 250 nM Ara-C, 600 µM HU and 30 µM haemin were incubated with FITC-conjugated NucView caspase-3 substrate and PE-conjugated anti-GPA monoclonal antibodies and analysed by flow cytometry (FL1 and FL2 channels respectively). In selected experiments, the caspase-3 inhibitor zDEVD-fmk (50 µM) was added to the cultures 30 min prior to drug applications and incubated with drugs for 4 days. Density plots are from a single typical experiment. The percentage of caspase-3-positive (B), GPA-positive (C) and GPA/caspase-3-double-positive (D) cells were determined from density plots obtained in drug-treated cultures with or without zDEVD-fmk. (E) K562 cells were treated with tested drugs at selected concentrations for 4 days with or without zDEVD-fmk (50 µM). Next, differentiation was assessed by benzidine staining. Data represent the mean ± SD of at least three independent experiments carried out in triplicate. Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) for the data points obtained with versus without zDEVD-fmk. Ara-C, cytosine arabinoside; cisPt, cisplatin; DOX, doxorubicin; FITC, fluorescein isothiocyanate; GPA, glycophorin A; HU, hydroxyurea; PE, phycoerythrin; PI, propidium iodide.

Figure 4

Mitochondrial transmembrane potential (ΔΨ_m) dissipation is observed in cells treated with DOX, cisPt, Ara-C, HU and haemin. Cells were cultured in the presence of 100 nM DOX, 5 µM cisPt, 250 nM Ara-C, 600 µM HU and 30 µM haemin for 3, 4 and 5 days at indicated concentrations. After treatment, cells were loaded with TMRE and analysed by flow cytometry (FL2 channel). (A) Histograms are shown from a single typical experiment. (B) Quantitative data are presented as means ± SD of percentages of cells with low TMRE fluorescence intensity (<2 × 10²) from at least three independent experiments. Significant differences (P < 0.001) from the control data points were obtained, except for haemin on days 3 and 4 (P > 0.05). Ara-C, cytosine arabinoside; cisPt, cisplatin; DOX, doxorubicin; HU, hydroxyurea; TMRE, tetramethylrhodamine ethyl ester.

Figure 5

EMSA comparing the level of GATA-1-binding activity in nuclear extracts of untreated and DOX-, HU-, Ara-C-, cisPt- and haemin-treated K562 cells. EMSA was performed after incubation of ³²P-labelled GATA-1 ds-oligonucleotides with nuclear extracts as described in Methods. Competition experiments showing the specificity of the protein–DNA complexes are included: lane 1 contains unrelated ds-oligonucleotide denoted as ‘ns’, and lane 2 contains specific competitor (non-radioactive GATA-1 ds-oligonucleotide) denoted as ‘s’. The position of DNA–GATA-1 complexes and the free DNA probe are indicated by arrows. Quantitative results from three independent experiments showing band intensity obtained with nuclear extracts from drug-treated versus control cells are presented in the Results section. Ara-C, cytosine arabinoside; cisPt, cisplatin; DOX, doxorubicin; EMSA, electrophoretic mobility shift assay; HU, hydroxyurea.

Figure 6

A hypothetical model of caspase-3 involvement in pharmacologically-induced erythroid differentiation of K562 cells. Cytostatic drugs trigger activity of transcription factor GATA-1 (Figure 5; Jeannesson et al., 1997; Chénais, 1998; Partington and Patient, 1999; Gillet et al., 2002; Szulawska et al., 2007). Caspase-3 activity is simultaneously induced via mitochondrial pathway (Figure 4; Czyz et al., 2008). However, an elevated level of Bcl-X_L in K562 (Benito et al., 1996) can prevent the massive release of apoptotic molecules from mitochondria to the cytosol. High endogenous levels of chaperone protein Hsp70 (Guo et al., 2005) can protect GATA-1 and Bcl-X_L from caspase-3-mediated proteolysis. In turn, GATA-1 can promote expression of erythroid and anti-apoptotic genes (Weiss and Orkin, 1995; Szulawska et al., 2007). A pharmacologically-induced differentiation is, therefore, similar to normal erythropoiesis, also involving the GATA-1, Bcl-X_L and Hsp70 proteins (Ribeil et al., 2007), but as the Bcl-X_L and Hsp70 proteins are overexpressed in K562, erythropoietin is not needed to trigger the signalling cascades leading to the differentiation of erythroleukaemic cells. Thus, both proteins Bcl-X_L and Hsp70, which are expressed in K562 cells at high levels, might be important for keeping cells on track of differentiation in the presence of drug-activated caspases. The importance of Bcl-X_L is also supported by the observation that inhibition of Bcr-Abl by STI571 induces apoptosis in CML cells by down-regulation of Bcl-X_L (Oetzel et al., 2000). Grey boxes – changes induced by cytotoxic drugs; black boxes – proteins already present at high levels in K562 cells. ΔΨ_m, mitochondrial transmembrane potential; Bcl-X_L, Bcl-2-related gene, long isoform; CML, chronic myelogenous leukaemia; Hsp70, heat shock protein 70.