738 Vol. 9, 738–742, February 2003 Clinical Cancer Research Caspase 3 in Breast Cancer Norma O’Donovan, John Crown, Helen Stunell, progression. Secondly, most cytotoxic anticancer agents cause Arnold D. K. Hill, Enda McDermott, tumor regression, at least in part, by inducing apoptosis. There- Niall O’Higgins, and Michael J. Duffy1 fore, defects in apoptosis may cause drug resistance and result in treatment failure. Departments of Medical Oncology [N. O’D., J. C.] and Nuclear Although multiple genes are involved in apoptosis, the key Medicine [M. J. D.], St. Vincent’s University Hospital, and Department of Surgery [H. S., A. D. K. H., E. M., N. O’H., M. J. D.] mediators of the process are the caspases. Caspases are aspar- and Conway Institute of Biomolecular and Biomedical Science tate-specific cysteine proteases, which cleave their substrates on [A. D. K. H., M. J. D.], University College Dublin, Dublin 4, Ireland the carboxyl side of the aspartate residue (3, 4). Currently at least 14 different caspases are known to exist, of which two- thirds play a role in apoptosis. The caspases involved in apo- ABSTRACT ptosis can be divided into two main groups, the initiator Purpose: An inability to undergo apoptosis is widely caspases (e.g., caspases 8, 9, and 10) and the downstream thought to contribute to both tumorigenesis and tumor pro- effector caspases (e.g., caspases 2, 3, 6, and 7). It is the members gression. One of the key mediators of apoptosis is the thiol of the latter group that degrade multiple cell proteins and are protease caspase 3. In this investigation, caspase 3 mRNA responsible for the morphological changes in apoptosis. and protein expression in breast cancer was examined. Caspase 3 is the most widely studied of the effector Experimental Design: Caspase 3 was measured at the caspases. It plays a key role in both the death receptor pathway, mRNA level using reverse transcription-PCR and at the initiated by caspase 8, and the mitochondrial pathway, involving protein level using both Western blotting and activity assays. caspase 9. In addition, several studies have shown that caspase Levels of apoptosis were determined using an ELISA, which 3 activation is required for apoptosis induction in response to detects nucleosomes released during DNA fragmentation. chemotherapeutic drugs e.g., taxanes, 5-fluorouracil, and doxo- Results: Relative levels of caspase 3 mRNA were similar rubicin (5–8). Caspase 3 is produced as an inactive 32-kDa ؍ ؍ in breast carcinomas (n 103), fibroadenomas (n 25), proenzyme, which is cleaved at an aspartate residue to yield a ؍ and normal breast tissues (n 6). However, levels of both 12-kDa and a 17-kDa subunit. Two 12-kDa and two 17-kDa the precursor and active forms of caspase 3 were signifi- subunits combine to form the active caspase 3 enzyme. Caspase cantly higher in carcinomas compared with both fibroade- 3 cleaves a wide range of cellular substrates including structural ؍ ؍ nomas (P 0.0188) and normal breast tissues (P 0.0002). proteins (e.g., lamins) and DNA repair enzymes [e.g., poly- Levels of apoptosis were also highest in the carcinomas and (ADP-ribose) polymerase]. It also activates an endonuclease ؍ correlated significantly with active caspase 3 levels (r caspase-activated DNAse, which causes the DNA fragmentation ؍ 0.481; P 0.0003). In the carcinomas, expression of caspase that is characteristic of apoptosis (3). 3 showed no significant relationship with either tumor size, Compared with other genes involved in apoptosis (e.g., p53 tumor grade, nodal status, or steroid receptor status but was and the Bcl-2 family), relatively little work has been carried out significantly higher in ductal carcinomas than in lobular on caspase expression in breast cancer. The aim of this study ؍ carcinomas (P 0.0188). was therefore to investigate caspase 3 expression and its rela- Conclusions: We conclude that rates of apoptosis as tionship to apoptosis in breast cancer. measured by both caspase 3 activation and nucleosome re- lease are higher in breast cancer than in nonmalignant breast tissue. This finding would appear to conflict with the MATERIALS AND METHODS widely held belief that apoptosis is reduced in malignancy. Sample Processing. Primary breast carcinoma, breast fi- The proliferation:apoptosis ratio, however, may be higher in broadenoma, and normal breast tissue samples were obtained at carcinomas than in the corresponding normal tissue. the time of surgery. Before processing, necrotic tissue was removed. The tissue samples were then snap frozen in liquid INTRODUCTION nitrogen and stored at –80°C. Tissue samples were homoge- nized using a Mikro-Dismembrator U (Braun Biotech Interna- Apoptosis or programmed cell death is important in ma- tional, Melsungen, Germany), to yield a fine powder. An aliquot lignancy for two reasons (1, 2). Firstly, suppression of apoptosis of the powder was extracted with 50 mM Tris buffer (pH 7.4) appears to be a critical event in both cancer initiation and 2 containing 1 mM monothioglycerol and assayed for ER and PR by ELISA (Abbott Diagnostics, North Chicago, IL). The cutoff point for ER and PR was 250 fmol/g wet weight tissue. Received 3/23/02; revised 9/10/02; accepted 10/9/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: ER, estrogen receptor; PR, progesterone 1 To whom requests for reprints should be addressed. Phone: 353-1- receptor; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3- 2094607; Fax: 353-1-2696018; E-mail: [email protected]. phosphate dehydrogenase; AFC, aminotrifluoromethyl-coumarin. Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 739 Table 1 Levels and frequency of caspase 3 mRNA expression in normal breast tissue, fibroadenomas, and primary breast carcinomas, as determined by semiquantitative RT-PCR Tissue type n Mean Median % positive Normal 6 0.201 0.225 83.3 Fibroadenoma 25 0.212 0.163 56.0 Primary carcinoma 103 0.253 0.225 72.8 Values are expressed in arbitrary absorbance units, relative to GAPDH. RNA Extraction. RNA extractions were performed us- ing an RNace Total Pure kit (Bioline, Randolph, MA). RNA Fig. 1 Western blot analysis to measure expression of procaspase 3 (32 integrity was assessed by gel electrophoresis, and concentrations kDa) and the large subunit of active caspase 3 (17 kDa). Measurement ␤ were measured spectrophotometrically. of -actin was used to control for equal loading of protein samples. RT-PCR Analysis. cDNA was synthesized from 1 ␮gof ␮ total RNA, using 50 M oligo(dT)12–18 primers (Promega, Mad- ison, WI), 0.4 mM deoxynucleotide triphosphates (Promega), 1ϫ Moloney murine leukemia virus buffer, and 100 units of dopropyl)dimethylammonio]-1-propanesulfonic acid, and 10% Moloney murine leukemia virus reverse transcriptase (Invitro- sucrose (pH 7.2)] at 37°C for 100 min. Fluorescence readings gen, Carlsbad, CA). PCR was performed using primers for the were measured on a Fluoroskan Ascent FL (Thermo Lab- housekeeping gene GAPDH and caspase 3 (Maxim Biotech, San systems, Helsinki, Finland) using an excitation wavelength of Francisco, CA). Briefly, the 25-␮l reaction mix contained 5 ␮l 390 nm and an emission wavelength of 510 nm. A standard of cDNA, 250 ␮M deoxynucleotide triphosphates, 1ϫ multiple curve was constructed using AFC, and caspase 3-like activity polymerase chain reaction primers, 1ϫ multiple polymerase values were expressed as nmol product formed (AFC)/mg pro- chain reaction buffer, and 1.25 units of Taq polymerase (Pro- tein/min. mega). Amplification was performed using 2 cycles of 1 min at Apoptosis ELISA. Levels of apoptosis were measured in 95°C and 4 min at 56°C followed by 28 cycles of 1 min at 94°C tumor samples using the Cell Death Detection ELISA (Roche, and 2.5 min at 56°C, with a final extension of 10 min at 72°C. Mannheim, Germany), which detects nucleosome fragments PCR products were visualized on a 2% agarose gel. Band released into the cytoplasm during apoptosis (9, 10). In brief, 30 ␮ intensities were measured by densitometry using the Eagle Eye g of cytosolic extracts, in duplicate, were incubated in strepta- gel documentation system (Stratagene, La Jolla, CA) and ex- vidin-coated microtiter plates with the biotin-conjugated anti- pressed as arbitrary units, relative to GAPDH. histone antibody and the peroxidase-conjugated anti-DNA anti- Caspase 3 Western Blotting. Cytosolic protein extracts body for2hatroom temperature, followed by washing and were prepared by extraction in Tris (pH 7.4) containing 0.1% colorimetric detection using ABTS substrate. Absorbances were Triton X-100. Protein concentrations were determined using the measured at 405 nm (reference wavelength, 490 nm). A stand- BCA protein assay (Pierce, Milwaukee, WI). Fifty ␮g of protein ard curve was constructed using the nucleosome solution sup- from each sample were separated on a 12% polyacrylamide gel. plied with the kit. Apoptosis values were expressed as concen- ␮ After electrophoresis, protein was transferred to a nitrocellulose tration of nucleosomes ( g/mg total protein). membrane that was blocked for1hatroom temperature in 1ϫ Statistical Analysis. Nonparametric Spearman rank cor- ␹2 Tris-buffered saline containing 5% skimmed milk powder, 1% relations, Mann-Whitney t tests, and tests were performed Ͻ BSA, and 0.05% Triton X-100. The membrane was then incu- using StatView 5.0.1. (SAS Institute Inc., Cary, NC). P 0.05 bated overnight at 4°C in the above blocking solution containing was regarded as statistically significant. rabbit anti-caspase 3 polyclonal antibody (1:1000; BD Trans- duction Laboratories, San Diego, CA).