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Granzyme A mediates glucocorticoid-induced apoptosis in leukemia cells¹

MASATERU YAMADA^*,, AKIRA HIRASAWA^*, SATOSHI SHIOJIMA^* and GOZOH TSUJIMOTO^*,2

^* Department of Molecular, Cell Pharmacology, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya, Tokyo 154-8567, Japan; and
Genox Research, Inc., Teikyo University Biotech Center, 907 Nogawa, Miyamae, Kawasaki 216-0001, Japan

²Correspondence: Department of Molecular, Cell Pharmacology, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya, Tokyo 154-8567, Japan. E-mail: gtsujimoto{at}nch.go.jp

SPECIFIC AIMS

The ability of glucocorticoids to induce apoptosis in lymphoid cells has resulted in their widespread use as chemotherapeutic agents for various leukemias, but the molecular mechanisms responsible for their apoptotic effects are uncertain. Based on a cDNA microarray analysis of 697 leukemia cells, a glucocorticoid-sensitive human pre-B acute lymphocytic leukemia cell line, we examined the function of granzyme A in glucocorticoid-induced apoptosis.

PRINCIPAL FINDINGS

1. Dexamethasone-induced apoptosis in 697 leukemia cell
697 leukemia cells were treated with clinically relevant concentrations of 100 nM dexamethasone for various periods and the numbers of viable cells were detected by the Trypan blue dye exclusion test. After 48 h of dexamethasone treatment, there was a decrease in the number of viable cells. Gel electrophoresis of DNA prepared from cells 36 h after dexamethasone treatment displayed DNA fragmentation indicative of apoptotic chromatin damage. The activity of caspase-3 in the cells increased after 6 h of dexamethasone treatment and continued to increase until 24 h. These results indicated that dexamethasone induces apoptotic cell death in 697 cells.

2. Expression of dexamethasone-regulated genes
To investigate gene expression during the dexamethasone-induced apoptotic process in 697 cells, cDNA microarrays analysis was performed. Twenty-six genes were identified that were differentially regulated in response to dexamethasone. The expression of 17 genes including granzyme A, glucocorticoid receptor, and suppressor of cytokine signaling (SOCS) -2 were up-regulated, whereas the expression of 9 genes including early growth response 1 and acute myeloid leukemia 1 were down-regulated in cells treated with dexamethasone for 24 h. To test the reliability of the above data, total RNA prepared from cells treated with dexamethasone was subjected to RT-PCR analysis, which confirmed the induction of genes such as granzyme A, glucocorticoid receptor, and SOCS-2.

3. Expression of dexamethasone-regulated genes in 697 leukemia cells stably transfected with Bcl-2
To circumvent the problem of recovering mRNA from dying cells, 697 cells stably infected with a retrovirus encoding the anti-apoptotic protein Bcl-2 were used, establishing the cell line 697-BCL2. It was confirmed that overexpression of the Bcl-2 protein in the 697-BCL2 cells rendered leukemia cells highly resistant to apoptotic cell death induced by dexamethasone. Total RNA prepared from 697 cells and 697-BCL2 cells treated with dexamethasone were subjected to RT-PCR analysis, and the expression of dexamethasone-regulated genes such as granzyme A, glucocorticoid receptor, and SOCS-2 were examined. No significant differences in the expression of these genes in 697 cells and 697-BCL2 cells were observed. This suggests that changes in expression of these genes do not represent death-associated artifacts, but rather are specific to dexamethasone-induced apoptotic processes.

4. Induction of granzyme A during dexamethasone-induced apoptosis
Among the differentially expressed genes identified by microarray analysis, the functional role of granzyme A in glucocorticoid-induced apoptosis was examined further. RT-PCR analysis of total RNA extracted from dexamethasone-treated 697 cells was performed for various periods. After 6 h of dexamethasone treatment, levels of granzyme A mRNA increased and continued to increase until 24 h (Fig. 1 A, B). Corresponding well with an increase in mRNA, dexamethasone also led to increased granzyme A protein levels in a time-dependent manner (Fig. 1C ). The induction of granzyme A protein by dexamethasone was also confirmed in 697-BCL2 cells (Fig. 1D ). To examine whether the granzyme A protein induced by dexamethasone had enzymatic activity, the activity of granzyme A was monitored. The granzyme A activity in dexamethasone-treated cells was significantly (P<0.01) higher than that in untreated cells, indicating that dexamethasone induces an increase in granzyme A activity.

View larger version (42K): [in this window] [in a new window]   Figure 1. Induction of granzyme A mRNA and protein by dexamethasone in 697 cells and 697-BCL2 cells. Cells were incubated in the presence (+, •) or absence (–, O) of 100 nM dexamethasone (DEX) at 37°C for the indicated periods, then subjected to assay. A) Total RNA was extracted from 697 cells; 1 µg RNA was reverse transcribed and cDNA was amplified in a PCR reaction using specific primers for granzyme A and for G3PDH as an internal control. B) Induction of granzyme A mRNA by dexamethasone in 697 cells was quantified by densitometric analysis relative to G3PDH levels. C) Contents of granzyme A protein in 697 cells were determined by enzyme immunoassay. D) Contents of granzyme A protein in 697 cells and 697-BCL2 cells treated with dexamethasone for 24 h were determined with enzyme immunoassay. The means and SE of 4 independent experiments are shown. **P < 0.01 compared with untreated cells (Student's t test).

5. Granzyme A inhibitor 3,4-DCI blocks dexamethasone-induced apoptosis
To explore whether an enhanced activity of granzyme A is causally related to the dexamethasone-induced apoptosis of 697 cells, the effects of the granzyme inhibitor 3,4-dichloroisocoumarin (3,4-DCI) were examined further. The cell extracts prepared from dexamethasone-treated cells were incubated with 3,4-DCI (1, 2, and 5 µM) and the activity of granzyme A and caspase-3 was measured. The granzyme A activity was inhibited by 3,4-DCI in a concentration-dependent manner. On the other hands, the caspase-3 activity was not inhibited by 3,4-DCI. To study the effects of 3,4-DCI on the glucocorticoid signaling, RT-PCR analysis of total RNA extracted from cells treated with dexamethasone and 3,4-DCI was performed. 3,4-DCI (1-5 µM) had no effect on the dexamethasone-induced increase in mRNA levels for granzyme A, glucocorticoid receptor, and SOCS-2. To study the anti-apoptotic effect of 3,4-DCI, cells were treated with dexamethasone and 3,4-DCI, and the activity of caspase-3 in the cells was measured. Dexamethasone-induced caspase-3 activity was markedly inhibited by cell treatment with 3,4-DCI in a concentration-dependent manner, showing that the inhibition of granzyme A rendered the leukemia cells highly resistant to dexamethasone-induced apoptosis (Fig. 2 A). The effect of granzyme A inhibition on apoptosis was assessed by FITC-annexin V staining; 3,4-DCI also suppressed the dexamethasone-induced annexin V staining (Fig. 2B ).

View larger version (24K): [in this window] [in a new window]   Figure 2. Inhibition by 3,4-DCI of dexamethasone-induced apoptosis in 697 leukemia cells. A) Caspase-3 activity. 697 leukemia cells were incubated in the presence or absence of 100 nM dexamethasone (DEX) and with various concentrations of 3,4-DCI at 37°C for 24 h. Cells were lysed and incubated with the substrate Ac-DEVD-AMC, then analyzed by a fluorescence microreader. To compare independent experiments, the basal activity in untreated cells was arbitrarily defined as 1 unit. B) Annexin V staining. 697 leukemia cells were incubated in the presence or absence of dexamethasone (DEX, 100 nM) and 3,4-DCI (5 µM) at 37°C for 6 h, labeled with FITC-annexin V, then subjected to flow cytometry. Cells that labeled positively for annexin V were considered to be apoptotic. The data are given as percentages of cells that were apoptotic. The means and SE of 4 independent experiments are shown. **P < 0.01 compared with cells not treated with 3,4-DCI (Dunnett's test).

CONCLUSIONS AND SIGNIFICANCE

In this study, we analyzed the genes responsible for mediating apoptotic effects of glucocorticoid on leukemia cells and identified the genes that are regulated in 697 cells in response to dexamethasone treatment. Comparison of our cDNA microarray analysis with a previously published report with an oligonucleotide array showed a large overlap of genes. Among genes differentially expressed after dexamethasone treatment, granzyme A is especially intriguing.

Granzymes are serine proteases and effector molecules, and are packaged in cytotoxic granules of cytotoxic T lymphocytes and natural killer cells, together with the pore-forming protein perforin. The concerted action of these molecules induces apoptosis of target cells, such as infected cells or transformed tumor cells. However, in nontarget cells, including leukemia cells, their precise function remains unclear. If glucocorticoid-regulated granzyme A activity is correlated with apoptosis, the up-regulation of granzyme A might facilitate the apoptosis induced by glucocorticoid in leukemia cells. In the present study, we examined this possibility and provided evidence that dexamethasone induces up-regulation of granzyme A and that this up-regulated granzyme A is involved in apoptosis induced by dexamethasone in 697 cells. This conclusion is based on the following observations: 1) exposure to dexamethasone increased the mRNA and protein levels of granzyme A in 697 cells (Fig. 1A-C ); 2) kinetics of the increase in mRNA and protein levels of granzyme A were consistent with an increase in caspase-3 activity in dexamethasone-treated cells; 3) exposure to dexamethasone increased the enzymatic activity of granzyme A in the cells; 4) the enzymatic activity of granzyme A was inhibited by the inhibitor 3,4-DCI in a cell-free system; 5) treatment with 3,4-DCI inhibited the dexamethasone-induced apoptosis in 697 cells (Fig. 2) ; 6) dose ranges of 3,4-DCI that inhibited dexamethasone-induced apoptosis were consistent with those that inhibited the enzymatic activity of granzyme A.

Overexpression of the Bcl-2 protein in 697-BCL2 cells rendered leukemia cells highly resistant to apoptotic cell death induced by dexamethasone treatment, although the levels of granzyme A mRNA and protein in the cells were increased (Fig. 1D ). These findings suggested that the Bcl-2 signaling terminates with the apoptotic signaling pathway mediated by granzyme A and that the granzyme A signaling induced by glucocorticoid acts upstream of Bcl-2 signaling in the apoptotic signaling pathway of glucocorticoid-treated leukemia cells. As to the substrate of granzyme A, it has been reported that the precursor of interleukin-1ß, putative HLA-associated proteins I and II, and lamins A, B, and C are substrates of granzyme A in vitro, although the relationships between these molecules and Bcl-2 signaling are unclear. Further investigation is necessary to clarify the mechanisms and consequences of granzyme A up-regulation in apoptotic signaling pathways induced by glucocorticoid in leukemia cells.

In conclusion, we have demonstrated that the gene expression of granzyme A was increased during dexamethasone-induced apoptosis in human leukemia 697 cells. Levels of granzyme A protein and its enzymatic activity in the cells were also increased by dexamethasone treatment. The inhibition of granzyme A activity inhibited dexamethasone-induced apoptosis. These results suggest that the induction of granzyme A is likely to play an important role in the control of apoptosis induced by glucocorticoid in leukemia cells.

View larger version (26K): [in this window] [in a new window]   Figure 3. Role of granzyme A in glucocorticoid-induced apoptosis in leukemia cells. Glucocorticoid increases expression and enzymatic activity of granzyme A leading to apoptosis in 697 leukemia cells. This pathway can be inhibited by either granzyme A inhibitor (3,4-DCI) or Bcl-2.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0116fje; doi: 10.1096/fj.02-0116fje

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