From the Institute of Molecular and Cell Biology, The National University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore
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ABSTRACT |
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Interleukin 1-converting enzyme-like proteases
(caspases) are crucial components of cell death pathways. Among the
caspases identified, caspase-3 stands out because it is commonly
activated by numerous death signals and cleaves a variety of important
cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the
requirement for caspase-3 in apoptosis, we took advantage of the MCF-7
breast carcinoma cell line, which we show here has lost caspase-3 owing
to a 47-base pair deletion within exon 3 of the CASP-3
gene. This deletion results in the skipping of exon 3 during
pre-mRNA splicing, thereby abrogating translation of the
CASP-3 mRNA. Although MCF-7 cells were still sensitive
to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no
DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features
typical of apoptotic cells such as shrinkage and blebbing. Introduction
of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or
staurosporine-induced apoptosis, it is required for DNA fragmentation
and some of the typical morphological changes of cells undergoing
apoptosis.
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INTRODUCTION |
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Apoptosis (programmed cell death) is typically accompanied by the activation of a class of death proteases (caspases) and widespread biochemical and morphological changes to the cell (1, 2). These changes almost invariably involve chromatin condensation and its margination at the nuclear periphery, extensive double-stranded DNA fragmentation, and cellular shrinkage and blebbing (3-5). However, apoptosis can also occur in the absence of DNA fragmentation (6-9). Recently, it has been demonstrated that a caspase activates an endonuclease (CAD)1 responsible for fragmentation of the DNA at the linker region between nucleosomes by specifically cleaving and inactivating ICAD (DFF45), the inhibitor of CAD (9-11).
There is evidence that caspases contribute to the drastic morphological changes of apoptosis by proteolysing and disabling a number of key substrates, including the structural proteins gelsolin, PAK2, focal adhesion kinase, and rabaptin-5 (12-15). The most commonly activated caspase (caspase-3) can mediate the limited proteolysis of these proteins, as well as the cleavage inactivation of DNA fragmentation factor (DFF45; ICAD) (9-11). Because there are several caspase-3-like proteases (1, 2), it is not known if caspase-3 is required in vivo for breakdown of DNA or cleavage of any of the proteins involved in maintaining cellular architecture. Here we show that MCF-7 carcinoma cells, which can be killed by apoptotic stimuli without DNA fragmentation and many of the hallmarks of apoptosis (7), are devoid of caspase-3 owing to a functional deletion in the CASP-3 gene. This has enabled us to address the question of whether caspase-3 is essential for double-stranded DNA breaks and some of the morphological alterations typical of apoptotic cell death.
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MATERIALS AND METHODS |
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Cell Lines, Reagents, and Antibodies-- The human Sy5y neuroblastoma cell line was provided by E. Feldman (University of Michigan), and the human breast carcinoma cell line MCF-7 was obtained from the ATCC. Cell lines were maintained in RPMI 1640 or Dulbecco's modified Eagle's medium (Sy5y) supplemented with 10% fetal calf serum, 10 mM glutamine, and 50 µg (each) of streptomycin and penicillin/ml (16). The origin and cultivation of HeLa D98 and H21 cells were as described (16).
The protease inhibitors aprotinin, bacitracin, antipain, leupeptin, and phenylmethylsulfonyl fluoride as well as staurosporine were purchased from Sigma. The specific activity of TNF was 4 × 107 units/mg protein. The monoclonal antibodies to CPP32 (caspase-3) were from Transduction Laboratories Inc. The monoclonal anti-actin antibodies were from Sigma.Preparation of Cell Extracts and Western Blotting-- Cell extracts were prepared as described (17). For detection of caspase-3, cell extracts were separated in 0.1% SDS, 12.5% polyacrylamide gels and subjected to Western blotting as described (16). The protein was visualized by the Amersham ECL kit.
cDNAs and RT-PCR--
The plasmid pcDNA3 containing the
full-length Yama (CASP-3) cDNA was provided by V. Dixit.
For the isolation of CASP-3 cDNA, total RNA was
extracted by the guanidinium thiocyanate method as described (16) or
with a Qiagen RNA purification kit reverse transcribed with the
SuperScript Kit (Life Technologies, Inc.) and amplified by PCR. The
following primers were used for the PCR (BamHI and
EcoRI sites are indicated by italicized letters): Y1,
5'-AAAGGATCCTTAATAAAGGTATCCATGGAGAACACT-3' (nucleotides 15 to +12 relative to the AUG in the CASP-3 mRNA); and Y5,
5'-AAAGAATTCTTAGTGATAAAAATAGAGTTCTTTTGTGAG-3' (nucleotides
+834 to +805 of CASP-3 mRNA). The PCR products were cloned into the BamHI/EcoRI sites of pUC18.
Cloning and Sequencing of Human CASP-3 Genomic
Fragments--
Genomic DNA was isolated from MCF-7 and Sy5y cells with
a Qiagen purification kit. Based on the genomic organization of the mouse CASP-3 DNA (18) and published cDNA sequences of
human CASP-3 (19), DNA fragments were amplified from both
cell lines by using two different primer sets specific for exons 2/3
(Y1/Y2) and exons 3/4 (Y3/Y4), respectively (see Fig. 2C).
Primer sequences (BamHI and EcoRI sites are
indicated by italicized letters) were: Y1,
5'-AAAGGATCCTTAATAAAGGTATCCATGGAGAACACT-3' (15 to +12:
exon 2); Y2, 5'-AAAGAATTCCAGTGCTTTTATGAAAATTCTTATTAT-3'
(+178 to +152: exon 3); Y3,
5'-AAAGGATCCAAAGATCATACATGGAAGCGAATCAAT-3' (+54 to +80:
exon 3); and Y4,
5'-AAAGAATTCCATCACGCATCAATTCCACAATTTCTT-3' (+307 to
+281: exon 4).
Determination of Cellular Sensitivity to TNF-- Apoptosis was induced with a combination of TNF (30 ng/ml) and cycloheximide (Chx; 10 µg/ml) or with staurosporine (1 µM) (Sigma). Cell death was measured with the standard TNF cytotoxicity assay as described (16).
Cell Cycle Analysis and DNA Fragmentation-- TNF-treated or untreated cells were fixed in ice-cold 80% ethanol, washed with phosphate-buffered saline and stained with propidium iodide (50 µg/ml, Sigma) at 37 °C for 60 min in the presence of RNase (20 µg/ml, Sigma) and 0.1% Triton X-100. Cell cycle analysis was performed using a Becton Dickinson FACScan. For each determination, a minimum of 20,000 cells were analyzed. For DNA fragmentation analysis, cellular DNA was prepared using the Blood and Cell Culture Mini DNA kit (Qiagen, Germany). Purified DNA was incubated for 2 h at 37 °C with 200 µg/ml RNase and analyzed on 1.6% agarose gels. DNA was visualized by ethidium bromide staining.
Stable Caspase-3 Transfection of MCF-7 Cells-- MCF-7 cells were stably transfected with caspase-3 or the expression vector alone (pcDNA3, Invitrogen) using the SuperFect Reagent (Qiagen, Germany). 40 h post-transfection, cells were trypsinized and reseeded in medium containing 800 µg/ml G418 (Life Technologies, Inc.).
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RESULTS AND DISCUSSION |
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Apoptosis Induced by TNF or Staurosporine Does Not Correlate with the Activation of Caspase-3-- HeLa D98 and H21 carcinoma cells, which are highly or only marginally sensitive to TNF-induced apoptosis, respectively (16), were treated with TNF or staurosporine, and the percentage of apoptosis was compared with the activation of pro-caspase-3. Because the caspase-3 antibody used for Western blot analysis did not detect the active 17-kDa subunit, the activation/processing of caspase-3 was judged by the disappearance of the 32-kDa precursor form. A 4-h treatment with TNF/Chx killed 78% of HeLa D98 cells (Fig. 1A) and resulted in a complete loss of the 32-kDa caspase-3 precursor form (Fig. 1B, compare lanes 1 and 2). In contrast, pro-caspase-3 was efficiently processed in HeLa H21 cells following a 4-h treatment with TNF/Chx (Fig. 1B, lane 5), but only 18% of the cells were killed (Fig. 1A). There was also no correlation between the extent of cell death and activation of caspase-3 when both HeLa cell lines were exposed to the apo-ptosis inducer staurosporine (20). Although both cell lines were efficiently killed after a 16-h staurosporine treatment (Fig. 1A), extensive processing of pro-caspase-3 was observed only in HeLa D98 cells (Fig. 1B, compare lanes 3 and 6). These results suggest that TNF and staurosporine may activate different apoptotic pathways and that caspase-3 activation is dependent not only on the death stimulus but also on the cell type.
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Loss of Pro-caspase-3 in MCF-7 Cells Is Due to a Deletion in the CASP-3 Gene-- To investigate why pro-caspase-3 was absent in MCF-7 cells, Northern blot analysis was performed with total RNA from MCF-7, HeLa D98, and H21 cells, as well as Jurkat cells that were shown to express functional CASP-3 mRNA (19). All cell lines showed similar expression of the CASP-3 mRNA, and no apparent size differences were observed (data not shown). Therefore, the lack of pro-caspase-3 in MCF-7 cells is not due to the failure to transcribe CASP-3 mRNA.
To determine whether mutation(s) led to the loss of pro-caspase-3, total RNA prepared from MCF-7 cells was reverse transcribed and used for PCR amplification. The RT-PCR performed with specific primers flanking the entire coding region of the CASP-3 mRNA gave rise to a fragment of around 750 bp (Fig. 2A, lane 2), which was shorter than the expected 867 bp observed in two control reactions performed with total RNA from HeLa D98 and Jurkat cells (Fig. 2A, lanes 3 and 4). Sequence analysis revealed that the MCF-7 CASP-3 mRNA contained a deletion of nucleotides 54-178 (Fig. 2B), whereas the mRNA from HeLa D98 cells matched the published sequence of the CASP-3 cDNA (19). The deletion of 125 nucleotides found in the MCF-7 CASP-3 mRNA leads to a frame shift starting at codon 18, thereby creating a new in-frame stop codon 41 amino acids downstream of the initiation codon (Fig. 2B), thus explaining the lack of pro-caspase-3 in MCF-7 cells. Six clones from two independent PCRs all contained the same deletion, and no DNA corresponding to the full-length CASP-3 cDNA was detected, suggesting that the PCR did not lead to the introduction of mutations in this gene and that no wild-type allele of CASP-3 is expressed. Furthermore, several MCF-7 clones obtained from different sources were all found to contain the same deletion in the CASP-3 mRNA, implying that the loss of pro-caspase-3 is not due to clonal variation.
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Caspase-3 Is Required for DNA Fragmentation and Morphological
Changes of Apoptosis--
MCF-7 cells treated with various apoptotic
stimuli (e.g. transforming growth factor-1 or etoposide)
undergo cell death in the absence of DNA fragmentation (7). In
addition, the apoptotic stimuli TNF/Chx or staurosporine efficiently
killed MCF-7 cells (Fig. 1), but no DNA fragmentation was observed
(Fig. 3A). In contrast,
treatment of HeLa D98 cells with TNF/Chx or staurosporine resulted in
the appearance of the internucleosomal DNA laddering typical of cells
undergoing apoptosis (Fig. 3A, upper panel) (16). Recently, it has been reported that caspase-3 activates the
endonuclease CAD responsible for DNA fragmentation by specifically
cleaving and inactivating ICAD, the inhibitor of CAD (9, 10). However, because there are several caspase-3-like proteases, it is not known
whether caspase-3 is sufficient or essential for DNA fragmentation. To
investigate the hypothesis that caspase-3 is required for DNA fragmentation, CASP-3 cDNA was stably transfected into
MCF-7 cells, which resulted in the generation of 26 individual
caspase-3-expressing MCF-7 clones. In all of these clones, caspase-3
was not spontaneously activated, and there were no detectable
morphological changes (data not shown). However, the caspase-3
activation pathway was fully functional in these transfectants, because
TNF/Chx treatment of MCF-7.3.28 cells (one representative
caspase-3-expressing clone out of three tested) resulted in the
efficient activation of this protease as judged by Western blotting
(Fig. 3B, top panel). In addition, MCF-7.3.28
cells showed a slight increase in TNF sensitivity (data not shown), and
more importantly, DNA from TNF/Chx-treated MCF-7.3.28 cells exhibited
the internucleosomal DNA laddering typical of apoptotic cells (Fig.
3B, middle panel). No internucleosomal DNA
fragmentation was observed in MCF-7 cells transfected with the vector
alone (Fig. 3B, middle panel). Lighter exposures
of both agarose gels revealed extensive double-stranded DNA breaks as
judged by faster migrating DNA only in TNF/Chx- and
staurosporine-treated HeLa D98 cells (Fig. 3A, bottom
panel) and TNF/Chx-treated MCF-7 cells stably expressing caspase-3
(Fig. 3B, bottom panel). These data demonstrate
that the components required for DNA fragmentation are present and
fully functional in MCF-7 cells and, furthermore, indicate that
caspase-3 is essential for their activation.
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ACKNOWLEDGEMENTS |
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We thank P. Singh and C. Pallen for critically reviewing the manuscript, E. Feldman for providing the Sy5y neuroblastoma cell line, and V. Dixit for the CASP-3 expression vector.
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FOOTNOTES |
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* This work was funded by the Institute of Molecular and Cell Biology, National University of Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 65-874-3379;
Fax: 65-779-1117; E-mail: mcbrj{at}imcb.nus.edu.sg.
1 The abbreviations used are: CAD, caspase-activated DNase; ICAD, inhibitor of CAD; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); Chx, cycloheximide; TNF, tumor necrosis factor.
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REFERENCES |
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