Caspase-3 and Caspase-6 cleave STAT1 in leukemic cells

Signal Transducer and Activator of Transcription-1 (STAT1) is phosphorylated upon interferon (IFN) stimulation, which can restrict cell proliferation and survival. Nevertheless, in some cancers STAT1 can act in an anti-apoptotic manner. Moreover, certain malignancies are characterized by the overexpression and constitutive activation of STAT1. Here, we demonstrate that the treatment of transformed hematopoietic cells with epigenetic drugs belonging to the class of histone deacetylase inhibitors (HDACi) leads to the cleavage of STAT1 at multiple sites by caspase-3 and caspase-6. This process does not occur in solid tumor cells, normal hematopoietic cells, and leukemic cells that underwent granulocytic or monocytic differentiation. STAT1 cleavage was studied under cell free conditions with purified STAT1 and a set of candidate caspases as well as with mass spectrometry. These assays indicate that unmodified STAT1 is cleaved at multiple sites by caspase-3 and caspase-6. Our study shows that STAT1 is targeted by caspases in malignant undifferentiated hematopoietic cells. This observation may provide an explanation for the selective toxicity of HDACi against rapidly proliferating leukemic cells.


Quantitative real-time PCR
mRNA was isolated and cDNA was synthesized as explained [39]. Primers are listed in Table 1. Data obtained from qRT-PCR were verified with the geNorm program.

Mass spectrometry
Protein bands were prepared for MS-analysis by proteolytic digestion with trypsin or V8-protease (GluC) according to Shevchenko et al. [69]. A LC-MS -system consisting of an Ettan-MDLC™chromatography system (GE Healthcare, Munich, FRG) coupled to an ESI-ion trap masspectrometer (type LTQ, Thermo Electron, USA). Data analysis to identify STAT1-related tryptic-and GluCspecific peptides was performed with the Thermo Proteome Discoverer 1.0™ software ((Thermo Electron Corporation, Erlangen, Germany) the human.fasta protein database. For N-terminal sequencing of the processed STAT1 products the Procise 494A protein sequencer (Applied Biosystems, Foster City, CA, USA) was used. was incubated either with caspase-3 or caspase-6 (1 unit). As control served pan-caspase inhibitor Z-VAD-FMK (zVAD) as well as heat shock (hs) inactivating of caspase activity. Extracts were analyzed by SDS-PAGE and stained with Coomassie solution.

Supplemental
4.2 Potential cleavage sites within the STAT1 protein were changed. Asparagine was mutated to Alanine. WT-GST-STAT1 as well as single mutants were assayed with 1 unit either caspase-3 or caspase-6 for 3 hours at 37° C. Western blot analysis was performed by the usage of a STAT1 antibody.
4.3 Proteolytic processing in vitro of GST-STAT1 by caspase-3 and caspase-6. The experiments were performed in three independent experiments. Lane 1: Untreated GST-STAT1. A single step batch procedure with GSH-Sepharose was used to prepare the protein. Numerous protein bands below the mature GST-STAT1 protein with a molecular weight of approximately 120 kDa are seen. Selected bands were checked for protein identity by mass spectrometry. Obviously all of them represent GST-STAT1 degradation products that occurred during protein expression and/or purification. In contrast, no E.coli proteins were among them. Lanes 2 and 3: GST-STAT1 treated with caspase-3 and -6, respectively. Treatments were performed in caspase cleavage buffer for 6 h at 37 o C. This point in time was selected after evaluation of the kinetics of caspase processing (data not shown).
Mass spectrometry analyses were performed with selected bands, marked with arrows in Supplemental Figure 4.3. Comparison of the protein pattern suggests that only a limited number of protein bands in lanes 2 and 3 really refer to GST-STAT 1 protein processing products induced by treatments with caspase-3 and caspase-6. To determine the particular cleavage areas of caspases we assessed the N-terminal and C-terminal endpoints of the polypeptide products, i.e. the maximum span of the polypeptide chain. To this end we attempted to localize the tryptic-and V8-protease proteolytic peptides prepared from the protein bands within the GST-STAT1 sequence by mass spectrometry. As an alternative approach the N-termini were determined by N-terminal sequencing by Edman degradation after blotting the protein gels on PVDF membranes.
Unfortunately, for some selected bands no interpretable MS-and/or N-terminal sequencing results could be obtained. Therefore, we are able to present results for a restricted number of bands indicated in Supplemental Figure 4.3 with numbers 1 and 6 (caspase-3 treatment) and with numbers 11, 12 (caspase-6 treatment). The drawing below demonstrates the principle analytical approach with band 1 as an example: The partial amino acid sequence of the GST-tag including a linker sequence and the functional domains of STAT1 are highlighted by different colors as indicated in the legend. The N-terminus detected by sequencing is shown in bold blue letters; tryptic-and V8-peptides are shown in bold red and yellow letters, respectively Supplemental By N terminal sequencing the N-terminus of the GST-STAT1 was detected (MSPIL). The trypticpeptides detected by MS were found to be distributed nearly over the whole (remaining) protein sequence, while the V8-peptide VHPSRLQTTDNLLPMSPE was the most C-terminally located peptide. The conclusion is: Caspase-3-processind occurred in the polypeptide stretch that follows the V8-peptide, i.e. the caspase-3-cleavage site (area) is located in the transcriptional activation domain.
The calculated molecular weight of the protein fragment is about 112 kDa. Using this approach the caspase-3 and caspase-6 cleavages sites were located (narrowed, described) for all selected protein bands. The results are summarized in the below-mentioned table (Supplemental