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Carcinogenesis, Vol. 21, No. 1, 7-14, January 2000
© 2000 Oxford University Press

Cancer Biology

Involvement of p21Waf1/Cip1 and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate

F. Chai, A. Evdokiou1, G.P. Young2 and P.D. Zalewski3

Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011,
1 Department of Orthopaedics and Trauma, University of Adelaide, Royal Adelaide Hospital, Adelaide, South Australia 5000 and
2 Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5032, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Butyrate, a short chain fatty acid produced in the colon, induces apoptosis in cancer cell lines by a sequential process involving inhibition of histone deacetylase, de novo protein synthesis and activation of DEVD-caspase, a major effector of apoptotic DNA fragmentation and membrane blebbing. We now show, in LIM 1215 colorectal cancer cells, that butyrate, in addition to activating DEVD-caspase and inducing apoptosis, also increases expression and cleavage of the universal cyclin-dependent kinase inhibitor p21Waf1/Cip1 and leads to hypo-phosphorylation of retinoblastoma protein. Accompanying these molecular changes was a progressive loss of G0/G1 and S phase cells. Expression of p21 had similar kinetics to that of the essential protein required for DEVD-caspase activation, indicating parallel effects of butyrate on anti-apoptotic and pro-apoptotic mechanisms. LIM 1215 cells, which were resistant to butyrate-induced apoptosis, were selected by three cycles of exposure to butyrate and removal of floating apoptotic cells. These cells showed markedly enhanced p21 expression and were in cell cycle arrest as determined by flow cytometry. On the other hand, subsequent culture of these cells for 2–3 days in the absence of butyrate resulted in down-regulation of p21 and restoration of sensitivity to apoptosis by butyrate. Western blots of butyrate-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band (p15) that was not present in control cultures. This band became apparent immediately after the onset of DEVD-caspase activation, was enriched in the floating apoptotic cell population when compared with the adherent, non-apoptotic cells and was absent in butyrate-resistant cells lacking DEVD-caspase activity. Peptide caspase inhibitors partially blocked appearance of p15. Here we show, for the first time, that p21 is a target of effector caspases in colorectal cancer cells and that the resistance to butyrate-induced apoptosis is characterized by failure of p21 cleavage.

Abbreviations: CDK, cyclin-dependent kinase; CRC, colorectal cancer; FCS, fetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBSS, Hank's balanced salt solution; PBS, phosphate-buffered saline; Rb, retinoblastoma protein; zDEVD-AFC, z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; zDEVD-fmk, z-Asp-Glu-Val-Asp-fluoromethyl ketone; zVAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
There are a range of different strategies available for prevention of colorectal cancer (CRC) which involve intervening in the multistage process leading to invasive tumor cells (1). Colorectal tumorigenesis is strongly influenced by environmental factors, especially products of carbohydrate fermentation in the colonic lumen (2–4). CRCs may be exposed to these substances from the time the initial clone of cancer cells emerges. Of particular importance in protection are dietary polysaccharides (e.g. wheat bran) which exert their inhibitory effects at the aberrant crypt stage of tumorigenesis (5). The short chain fatty acid butyrate, a colonic fermentation product of dietary polysaccharides, partly mediates their protective action (6) and is thought to protect via modulation of expression of colonic epithelial genes involved in cell cycle arrest, differentiation and apoptotic cell death. Butyrate is a potent inducer of apoptosis in CRC and other cancer cell lines at physiological concentrations (7–9). Butyrate appears not to be toxic for normal colonocytes (4), perhaps because it is rapidly metabolized (as a fuel) by these cells (10).

One of the earliest cellular effects of butyrate is hyper-acetylation of histones resulting in altered patterns of gene expression. Hyper-acetylation, due to non-competitive inhibition of histone deacetylase, occurs within 30 min in vitro, as well as in vivo, where high fiber diets promote histone hyper-acetylation in colonic epithelia of rats (11). That histone acetylation triggers induction of apoptosis by butyrate is supported by our finding that trichostatin A, a more specific inhibitor of histone deacetylase, mimics butyrate in the kinetics of induction of apoptosis in colon cancer cells (9). Acetylation of core histones H3 and H4 weakens their association with DNA, thereby promoting transcription factor binding to regulatory elements of genes and enhancing gene expression (12,13). Included in this may be one or more cell death genes (which presumably are normally tightly repressed) since butyrate-induced apoptosis is blocked by inhibitors of RNA and protein synthesis (9).

Principal amongst the downstream effector molecules in apoptosis is the family of caspases which cleave certain critical targets, leading to either inactivation of the function of the protein or generation of a pro-apoptotic cleavage product (14–16). Cleavage occurs next to D (Asp) but different caspases may have different substrate specificities. Caspase-3 cleaves after a DXXD motif (X is any of a subset of amino acids) (17). In one cellular substrate, cleavage is next to DEVD and, therefore, a fluorogenic substrate assay for caspase-3 has been developed based on liberation of a leaving group AFC from DEVD-AFC (associated with an increase in green fluorescence). Although this has been used as a caspase-3-specific assay, it is now clear that caspases-2 and -7 may also cleave this substrate (17). For this reason, we will here use the less specific term DEVD-caspase. Induction of DEVD-caspase activity and caspase precursor processing in the cytoplasm of cells preceding morphological changes, suppression of this increase by cycloheximide and inhibition of butyrate-induced apoptosis by membrane-permeable inhibitors of caspases (9) indicate that butyrate-induced apoptosis involves activation of caspase-3 (and/or related caspases). Levels of caspase-3 proenzyme do not change in butyrate-treated cells (9) indicating that butyrate modulates a factor(s) required for caspase-3 activation.

The orderly progression of cells through different cell cycle states is controlled by the sequential activity of various cyclin-dependent kinases (CDKs) which are activated by cyclins and inhibited by CDK inhibitors (18). There are two major classes of CDK inhibitors, which exert control by binding to cyclin–CDK complexes: the CIP/KIP family, including p21Waf1/Cip1, p27Kip1 and p57Kip2, capable of interacting with most cyclin–CDK complexes, and a second family p15INK4B/MTS2, p16INK4/MTS1, p18 and p19, with a more restricted range of activity. The CIP/KIP family contain conserved cyclin-binding domains near their N-terminus where they bind to cyclin–CDK complexes; this interaction inhibits CDK kinase activity and hinders their ability to phosphorylate the retinoblastoma family of proteins (Rb). Increased levels of CIP/KIP proteins are associated with growth arrest and/or differentiation, although the particular protein involved appears to be cell type dependent. By far the most widely studied of these is p21Waf1/Cip1, a potent inhibitor of a wide array of CDK–cyclin complexes and which is responsible for G1/S arrest induced by p53 in response to DNA damage (18). p21 is also induced by p53-independent pathways (13). In addition to its N-terminal CDK inhibitory domain, p21 contains a PCNA-binding motif located in the C-terminal part of the protein which may be important for inhibition of DNA replication by p21 (19).

Butyrate is a potent inducer of p21 in CRC cells, even at sub-toxic concentrations (e.g. 1 mM), and p21 is required for butyrate-mediated growth arrest (13,20). However, in NIH 3T3 cells Vaziri et al. (21) reported that growth arrest in the G1 phase of the cell cycle in response to butyrate was largely independent of p21. Interestingly, p21 is associated with both induction of differentiation (22) and inhibition of apoptosis (23) in certain systems. Here we have studied the relationship of p21 expression to caspase activation and apoptosis in LIM 1215 CRC cells treated with butyrate, to investigate further the inter-relationships between butyrate induction of apoptosis and cell cycle arrest.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Major materials and their suppliers were: EDTA, EGTA, herring sperm DNA, Nonidet P-40 (NP-40), dithiothreitol, sucrose and HEPES (Sigma Chemical Co., St Louis, MO); sodium butyrate (BDH, Poole, UK); penicillin/streptomycin, EDTA/trypsin, glutamine and CHAPS (ICN, Aurora, OH); gentamicin (David Bull Laboratories, Melbourne, Australia); diphenylamine (Ajax, Auburn, Australia); z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-AFC), zVAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and z-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk) (Kamiya Biomedical Co., Tukwila, WA). All other reagents were reagent grade, unless indicated.

Cell cultures
LIM 1215 cells were cultured in HEPES-buffered RPMI 1640, pH 7.4 (ICN, Aurora, OH), supplemented with glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml), gentamicin (160 µg/ml) and 10% fetal calf serum (FCS) (Biosciences, Sydney, Australia) in a humidified atmosphere containing 5% CO2. Experiments were performed in either 25 cm2 vented tissue culture flasks or 6-well plates (Sarstedt, Newton, NC). LIM 1215 cells were allowed to attach and grow for 2–3 days prior to exposure to test reagents. For cultures with butyrate, stock solutions (400 mM) in RPMI were made fresh each day and diluted into the cell suspension to give the desired final concentration (in most experiments this was 1 or 4 mM).

Determination of morphological changes in apoptosis
For morphological assessment, cells were examined by phase contrast microscopy after addition of an equal volume of 0.2% trypan blue in Hank's balanced salt solution (HBSS), pH 7.4. Apoptotic cells were distinguishable from normal cells by their nuclear fragmentation, presence of apoptotic bodies, decreased size and, sometimes, intense membrane blebbing. Cells with one or more of these properties were scored as positive. In most cases, they excluded trypan blue. A minimum of 300 cells from replicate tubes were scored. In some experiments, acridine orange was added to a final concentration of 10 µM and cells analyzed by fluorescence microscopy for chromatin segregation.

Apoptotic DNA fragmentation
To assay DNA fragmentation, cells (5x106–107 in total) were lysed at 4°C in 1 ml of NP-40 lysis buffer (5 mM Tris–HCl, pH 7.5, containing 5 mM EDTA and 0.5% NP-40) and centrifuged at 13 000 g for 10 min at 4°C. Supernatant fractions containing low molecular weight DNA fragments were assayed for DNA by a fluorometric technique (24). Hoechst dye 33258 was dissolved in deionized water to a concentration of 1 mg/ml and stored at 4°C. Prior to use, 1 µl of dye was added per 10 ml of buffer (10 mM Tris–HCl, pH 7.4, containing 1 mM EDTA and 100 mM NaCl). Aliquots of lysate (20–50 µl) were placed into fluorimeter grade disposable cuvettes (Greiner) and 1 ml of diluted dye solution added. Fluorescence was measured at an excitation wavelength of 356 nm and emission wavelength of 458 nm (slit widths 10 nm) in a Perkin Elmer fluorescence LS50 spectrophotometer. Herring sperm DNA was used to derive a standard curve.

Measurement of DEVD-caspase activity
DEVD-caspase was assayed by cleavage of zDEVD-AFC, a fluorogenic substrate based on the peptide sequence at the caspase-3 cleavage site of poly(ADP-ribose) polymerase (14). Cells (5x106–107 per flask) were cultured with test reagents, washed once with 5 ml of HBSS and resuspended in 1 ml of NP-40 lysis buffer (as for DNA fragmentation). After 15 min in lysis buffer at 4°C, insoluble material was pelleted at 15 000 g and an aliquot of the lysate was tested for protease activity. To each assay tube, 20 µl of cell lysate was added, followed by 1 ml of protease buffer (50 mM HEPES, 10% sucrose, 10 mM DTT, 0.1% CHAPS, pH 7.4) containing 8 µM substrate. After 24 h at room temperature, fluorescence was quantified (excitation 400 nm, emission 505 nm) in a Perkin Elmer LS50 spectrofluorimeter. Optimal amounts of added lysate and duration of assay were taken from linear portions of curves as determined in preliminary experiments. One unit of caspase activity was taken as one fluorescence unit (at a slit width of 10 nm) per 24 h incubation with substrate.

Cell cycle analysis
Cells were removed from culture dishes by trypsinization, collected by centrifugation, resuspended in ice-cold phosphate-buffered saline (PBS) and then fixed in absolute methanol for at least 30 min. Cells were then washed in PBS containing 0.5% Tween 20, followed by two washes in PBS containing 2% FCS. After washing, cells were resuspended in PBS, 2% FCS containing 40 µg/ml RNase A and incubated for 20 min at 37°C. Finally, cells were washed in PBS, 2% FCS and resuspended in PBS containing propidium iodide (20 µg/ml). The stained nuclei were analyzed using a flow cytometer (Epics Profile; Coulter). Cell cycle distribution was based on 2N and 4N DNA content. Cells with less than 2N DNA content were indicative of apoptotic cells.

RNA extraction and northern blot analysis
For northern blot analysis, cells were seeded at a density of 3–5x105 cells/well, allowed to attach and grow for 2 days and incubated in the presence or absence of 4 mM butyrate. Total RNA was isolated at the indicated times using the Trizol Reagent (Gibco BRL) according to the manufacturer's instructions. Total RNA (10 µg/lane) was electrophoresed in formaldehyde–1% agarose gels, transferred to Hybond N+ nylon membranes (Amersham, Castle Hill, Australia) and immobilized by UV crosslinking. Membranes were pre-hybridized for 3 h at 42°C in 1 M NaCl, 1% SDS, 10% dextran sulfate, 50% formamide and 100 µg/ml heat-denatured herring sperm DNA. They were then hybridized with p21WAF1/CIP1 cDNA (a kind gift from Dr Helena Richardson, The University of Adelaide, Adelaide, Australia), which was radiolabeled with [{alpha}-32P]dCTP by random priming using the Giga Prime kit (Bresatec, South Australia). To allow quantification of mRNA signals, the same filters were reprobed with a 450 bp 32P-labeled PCR-generated DNA fragment of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Following hybridization for 16 h at 42°C, filters were washed at high stringency and resulting signals analyzed using a SF PhosphorImager (Molecular Dynamics Inc.).

Western blotting
Aliquots of cells were lysed in NP-40 lysis buffer (5 mM Tris–HCl, pH 7.5, containing 5 mM EDTA, 0.5% NP-40 and complete protease inhibitor cocktail; Boehringer Mannheim, Mannheim, Germany) and stored at –20°C until assayed. Protein concentration was measured using the DC protein assay kit (BioRad, Hercules, CA). One volume of 4x SDS loading buffer (250 mM Tris–HCl, pH 6.8, 40% v/v glycerol, 8% w/v SDS, 20% v/v 2-mercaptoethanol and 0.5% w/v bromophenol blue) was added to 3 vol of lysate and mixtures were denatured. Equal protein amounts (18–36 µg) were loaded into wells and electrophoresed on 7.5% minigels for Rb or 12% minigels for p21 (BioRad) at 200 V for 45 min. To ensure that equal amounts of protein were loaded in each track, duplicate gels were also stained with Coomassie brilliant blue G-250 (BDH, Sydney, Australia). Proteins were blotted onto Immobilon-P PVDF membrane (Millipore, Sydney, Australia). Membranes were preblocked with 3% powdered milk in Tris-buffered saline, 0.1% Tween-20, 3% bovine serum albumin for 60 min at room temperature. Primary antibodies used were mouse monoclonal antibodies against human p21 (Clone 6B6, 1/333 dilution; Pharmingen) and human Rb (1/333 dilution; Santa Cruz Laboratories) and polyclonal rabbit antibodies against human p27 (1/500 dilution; Calbiochem, Sydney, Australia). Secondary antibody was either sheep anti-mouse IgG peroxidase conjugate (1/1000 dilution, Fab fragment; Boehringer Mannheim) or donkey anti-rabbit Ig peroxidase conjugate (1/1000 dilution, whole antibody; Boehringer Mannheim). Primary antibodies were added for 60 min and secondary antibodies for 45 min at room temperature. Membranes were then soaked in ECL Western Blotting reagent (Amersham Life Sciences, Sydney, Australia) and bands detected by autoradiography. Rainbow high molecular weight markers (Amersham Life Sciences) were used for determination of molecular weight.

Experimental design
All experiments were repeated a minimum of three times. Typical experiments are described or data were pooled, as indicated in the text. Statistical significance was determined by Student's t-test and is indicated in the text or legends to figures.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Induction of growth arrest, apoptosis and DEVD-caspase activation in butyrate-treated LIM 1215 cells
Growth inhibition.
LIM 1215 cells were seeded into flasks and treated with physiologically relevant concentrations (0.5–8 mM) of sodium butyrate, 2 days after seeding. They were then assayed at daily intervals for 5 days (days 2–7 after seeding) for changes in adherent cell number (as a measure of viable cells). Figure 1AGo shows that growth was retarded at all concentrations of butyrate tested, but particularly at concentrations >=1 mM. Five days after addition of 4 or 8 mM butyrate, cell numbers were significantly lower than those at the time of addition of butyrate. Most of this decrease occurred in the first day after addition. Flow cytometric analysis with propidium iodide showed a progressive depletion of G1, S and G2 + M phase cells, within 30 h after addition of 4 mM butyrate (Figure 1BGo, left).



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  		Fig. 1. Induction of growth arrest and apoptosis of LIM 1215 cells by butyrate. (A) Growth curves. Cells (4x105) were seeded into wells at day 0 and varying concentrations of butyrate were added at day 2 (arrow). At daily intervals thereafter non-adherent cells were discarded and remaining adherent cells were harvested by trypsinization and counted. Control, {circ}; 0.5 mM butyrate, •; 1 mM butyrate, {triangleup}; 2 mM butyrate, {blacktriangleup}; 4 mM butyrate, {square}; 8 mM butyrate, {blacksquare}. The graph shows increasing growth arrest with increasing concentration of butyrate. A typical experiment is shown. Data are means of duplicates. (B) Cell cycle analysis following treatment with 4 mM butyrate. Butyrate (4 mM) was added at time 0 and at 3 h intervals combined non-adherent and adherent cell populations were washed, fixed and analyzed by flow cytometery (see Materials and methods). G1 cells, {circ}; S phase cells, •; G2 + M phase cells, {square}; `sub-G1' apoptotic cells, {blacksquare}. The graph shows decreases in cell number at all phases of the cell cycle and replacement by apoptotic cells, beginning after a lag period of ~7 h (left). Representative FACS scans at times 0, 6 and 21 h following butyrate treatment are shown on the right. Note the peak of apoptotic cells with less than 2N DNA content at 21 h treatment. (C) Cell cycle analysis following treatment with 1 mM butyrate. Butyrate (1 mM) was added as in (B) and the percentage of cells in each phase of the cell cycle determined over time. (Left) A significant increase in the number of cells in G1 and no evidence of apoptosis. Representative FACS scans at the indicated times are shown on the right. (D) DEVD-caspase activity and DNA fragmentation. Butyrate (4 mM) was added at time 0 and at 3 h intervals combined non-adherent and adherent cell populations were washed, lysed in NP-40 lysis buffer and assayed for DEVD-caspase activity (•) and DNA fragmentation ({circ}).

 
Apoptosis.
The decrease in cell numbers at higher concentrations of butyrate suggested that there was not only cell cycle arrest but also cell death. Flow cytometric analysis revealed an increase in apoptotic cells (filled squares in Figure 1BGo, left), as shown by the appearance of an increasing fraction of cells with less than 2N DNA content, preceding the G1 peak on the fluorescence scans (Figure 1BGo, right). In contrast, cells treated with 1 mM butyrate failed to undergo apoptosis and the number of cells in the G1 phase of the cell cycle increased significantly, 48 h post-butyrate treatment, from 59 to 73% (Figure 1CGo). These results suggest that growth inhibition of LIM 1215 cells by low concentrations of butyrate is associated with G1 arrest, whereas higher concentrations of butyrate such as 4 mM induce apoptosis. Examination of cells treated with 4 mM butyrate, by phase contrast microscopy, showed typical morphological features of apoptosis, including the presence of one or more intracellular apoptotic bodies, a granularity that was often localized to one pole of the cell, exclusion of the vital dye trypan blue, membrane blebbing and, in some cells, reduction in volume. Labeling with acridine orange revealed segmentation of the chromatin into discrete masses in the butyrate-treated cells, compared with the uniform mass of chromatin in untreated cells (not shown). Apoptosis was accompanied by detachment of the cells from the plates. In a typical experiment, incubation of cells for 24 h with 4 mM butyrate resulted in a detachment of 77% of the cells. This floating population consisted of 97% apoptotic cells as detected by morphological features while the adherent fraction contained only 12% apoptotic cells. There was also extensive DNA fragmentation, as measured by Hoechst dye labeling of DNA fragments (open circles in Figure 1DGo), comprising up to 36% of the total DNA.

Accompanying the above changes was activation of DEVD-caspase (filled circles in Figure 1DGo). There was little caspase activity in control cultures, but a marked increase beginning ~9 h after addition of butyrate and peaking at ~15 h. The time course of DEVD-caspase activation was similar to that for DNA fragmentation (Figure 1DGo) and apoptosis (Figure 1BGo). Although the bulk of DEVD-caspase activity was detected in the floating cell population, significant activity was also present in the adherent cells, indicating that activation of DEVD-caspase precedes loss of adhesion in butyrate-treated CRC cells.

Hypophosphorylation of Rb.
There was also a shift in the phosphorylation state of the cell cycle regulator Rb (Figure 2Go). Untreated cells showed the characteristic hypo-phosphorylated Rb and hyper-phosphorylated Rb forms. Disappearance of phosphorylated Rb was evident by 12–18 h after addition of butyrate, consistent with inhibition of CDK activity and cell cycle arrest. The apparent onset of dephosphorylation of Rb is coincident with detectable levels of caspase activation and apoptosis (Figure 1DGo).



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  		Fig. 2. Hypo-phosphorylation of Rb. Cells were treated for increasing times with 4 mM butyrate before western blotting with anti-Rb. The figure shows loss of slower-migrating hyper-phosphorylated pRb and accumulation of hypo-phosphorylated Rb, particularly between 12 and 18 h.

 
Reversible induction of butyrate resistance in LIM 1215 cells
Since exposure of LIM 1215 cells to butyrate resulted in death and detachment of only a proportion of the cells, those cells remaining attached (and viable) after 24 h with butyrate were subjected to a second and third cycle of treatment with butyrate. Figure 3AGo shows the result of a typical experiment (repeated four times) in which total DEVD-caspase activity (expressed per 105 cells) from both the adherent and floating cell populations was assayed. The initial level of DEVD-caspase activity was 3.0 ± 0.3 U caspase/105 cells. The cells were then incubated for 1 day with 4 mM butyrate. There was a substantial increase in DEVD-caspase activity (60.6 ± 5.5 U/105 cells) as shown in column Ad1. In the absence of butyrate, there were no changes in the levels of DEVD-caspase activity (2.7 ± 0.8 U/105 cells). After this first day, the floating cells in each well were decanted and the remaining adherent cells were cultured for a further 24 h with a second application of butyrate (4 mM). Results for this second addition of butyrate are shown in the column marked Ad2. In the presence of butyrate, there was still high activity of DEVD-caspase (38.7 ± 2.8 U/105 cells), although less than after the first day. The floating cells were again discarded and the remaining cells exposed to a third cycle of butyrate (Ad3). At this stage, there was negligible activation of DEVD-caspase (5.8 ± 0.8 U/105 cells). Therefore, in cultures exposed to three cycles of butyrate, there was selection for a butyrate-resistant subset of LIM 1215 cells.



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  		Fig. 3. Expression of p21 and cleaved p21 in butyrate-resistant LIM 1215 cells. (A) DEVD-caspase activity during induction of butyrate resistance. Cells were treated with or without 4 mM butyrate at 24 h intervals and DEVD-caspase activity measured on combined adherent and non-adherent cell populations, expressed per 105 cells. The figure shows that DEVD-caspase activity increases markedly on the first day of treatment with butyrate (Ad1) but is sequentially lost following three cycles of addition of butyrate. By day 3 (Ad3), the cells were resistant to butyrate-induced caspase activation. Resistance was reversed when cells were grown in the absence of butyrate for 2 days (W1 and W2), which resulted in sensitive cells when butyrate was added again following this withdrawal period (Ad1'). These changes were accompanied by growth arrest and apoptosis. (B) Western blots with anti-p21 for corresponding extracts. Only those from butyrate-treated cells are shown. The figure shows an increase in p21 expression during induction of resistance by butyrate (Ad2 and Ad3) and a decrease in p21 during the withdrawal period (W1 and W2). Note also the strong signal of cleaved p21 in Ad1 but not Ad1'. The latter had a weak band that is not evident on the photomicrograph. A densitometric profile showing the relative levels of p21 protein in each track is shown at the bottom. The results are representative of a typical experiment repeated three times.

 
To determine whether the resistance to butyrate was permanent, the cells were cultured in the absence of butyrate for 2 days. During this withdrawal period (columns W1 and W2 in Figure 3AGo), there was no significant DEVD-caspase activation over that in control cells. When butyrate was added after the withdrawal period (Ad1'), there was substantial activation of DEVD-caspase (58.2 ± 6.7 U/105 cells), similar to that obtained in the initial treatment with butyrate (Ad1), suggesting complete restoration of sensitivity to butyrate. Therefore, the resistance to butyrate in cells at the Ad3 stage was only temporary and disappeared when cells were cultured in the absence of butyrate for 2 days. Subsequent experiments showed that in order to reverse the resistance to butyrate at the Ad3 stage, butyrate had to be withdrawn for at least 2 days (data not shown).

Induction of p21Waf1/Cip1 by butyrate
One possibility was that resistance to butyrate-induced apoptosis was determined by a cellular protein whose expression was regulated by butyrate. Since butyrate is known to up-regulate the cell cycle regulator p21Waf1/Cip1 (13), induction of p21 in butyrate-treated LIM 1215 cells was determined by western blotting. Figure 3BGo shows a correlation between the levels of p21 protein and resistance to butyrate-induced apoptosis. Initially, the cells had moderate levels of p21 protein (lane Ad0). After addition of butyrate for 1 day (lane Ad1), there was a modest increase in p21, in addition to a lower molecular weight band, p15, which is likely to be a cleavage product (see below). After three cycles of addition of 4 mM butyrate and removal of floating cells, the remaining adherent cells, which were resistant to butyrate-induced apoptosis, had very high levels of p21 protein; the p15 band was no longer detectable (lane Ad3). When butyrate was withdrawn, there was a decline in the level of p21. It is interesting to note that the decline in p21 preceded reversal of resistance since at day 1 of withdrawal (lane W1), the cells were still resistant to addition of butyrate (data not shown) but p21 levels had declined. By day 2 of withdrawal of butyrate (W2), cells were now sensitive to butyrate but p21 levels were no lower than on day 1. This suggests a lag of at least 24 h between decline in p21 and re-acquisition of sensitivity to butyrate. When butyrate was re-added after the withdrawal period, the p21 level rose again and there was a faint band corresponding to p15 (track Ad1'). These data suggest that induction of p21 by butyrate in CRC cells retards entry into apoptosis.

To investigate further the relationship between p21 (and p15) expression and induction of apoptosis, the kinetics of expression were studied at the level of mRNA (Figure 4AGo) and protein (Figure 4BGo). Basal levels of p21 mRNA (detected by northern blotting) are shown at time 0 in Figure 4AGo. A substantial increase occurred within 3 h after addition of 4 mM butyrate and decreased thereafter, but remained elevated over basal levels up to 12 h. Beyond 12 h, p21 mRNA declined rapidly and was almost undetectable at 30 h. Equal loading of RNA in each track was confirmed by hybridization of the filters with a GAPDH control probe (Figure 4AGo, middle). The increase in p21 protein peaked at 9 h after addition of butyrate and was maintained at elevated levels up to, at least, 24 h (Figure 4BGo). As is evident from a comparison of Figures 1D and 4BGoGo, induction of p21 peaked at the time of onset of activation of DEVD-caspase.



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  		Fig. 4. Kinetics of expression of p21 and p15 in butyrate-treated cells. (A) p21 mRNA. Cells were treated with or without 4 mM butyrate for the indicated times and p21 mRNA assessed by northern blotting (top). The figure shows induction of p21 mRNA 3 h after addition of butyrate; this was maintained at high levels up to 12 h. Blots were rehybridized with a cDNA probe specific for GAPDH to indicate RNA loading (bottom). Results were analyzed by densitometry and expressed as a ratio of p21 to GAPDH mRNA (bar chart). (B) Western blot of p21 protein and cleaved p21 (p15). The figure shows induction of p21 protein beginning at 9 h followed by appearance of p15 at 15 h after addition of butyrate. A densitometric profile showing the relative levels of P21 protein in each track is shown at the bottom. The results are representative of three independent experiments. (C) Relative amounts of p15 and p21 in adherent and non-adherent cell populations. Cells were treated with or without 4 mM butyrate for 24 h and lysates analyzed by western blotting with anti-p21 antibody. (a) Untreated cells (combined non-adherent and adherent populations); (b) butyrate-treated cells (combined non-adherent and adherent populations); (c and d) butyrate-treated cells (apoptotic cell-rich non-adherent population); (e and f) butyrate-treated cells (viable or pre-apoptotic adherent population). The figure shows enrichment of p15 relative to p21 in apoptotic cells. Note the large decrease in the absolute amount of p21 fragment in apoptotic cells suggesting that once cleaved, p21 may undergo rapid degradation.

 
Cleavage of p21 is associated with entry into apoptosis in butyrate-treated LIM 1215 cells
The p15 band appeared at time points beyond 12 h following addition of 4 mM butyrate and peaked at 24 h (Figure 4BGo). This band was consistently seen in western blots of p21 in butyrate-treated LIM 1215 cells but varied considerably in intensity. Its first appearance soon after induction of DEVD-caspase in these cells (compare Figure 1DGo) suggested it may be a consequence of proteolytic cleavage of p21 by DEVD-caspase or another member of the caspase family. In support of this hypothesis, when lysates of the floating non-adherent (apoptotic-rich) cell population from 24 h butyrate-treated samples were run individually on western blots, p15 was the predominant fragment (Figure 4CGo, lanes c and d). This suggests that p21 is entirely cleaved late in apoptosis of CRC cells. On the other hand, when the lysates of adherent (largely viable or pre-apoptotic) cells were run, both p21 and p15 were detected (Figure 4CGo, lanes e and f), implying that p21 is cleaved prior to detachment of the cells from the substratum. Lysates of adherent cells contained significant DEVD-caspase activity (not shown). Note that, despite loading equivalent amounts of protein in lanes c–f, the p15 band in the lysates of the apoptotic population was much less intense than that of the viable cell population. This may indicate that p15 is degraded further, later in apoptosis. The absence of p15 in lysates of butyrate-resistant cells, which lacked DEVD-caspase activity (Figure 3BGo, lane Ad3) is consistent with the hypothesis that p15 is derived from p21 by caspase cleavage. Somewhat surprisingly, only a very faint p15 band was seen when butyrate was added to these cells after the 2 day withdrawal period (track Ad1'), despite these lysates having equivalent amounts of DEVD-caspase activity to those in track Ad1.

Further evidence for a relationship between p15 and caspase cleavage was sought using peptide caspase inhibitors. LIM 1215 cells were pre-treated with either of two peptide inhibitors, the more specific DEVD-caspase inhibitor zDEVD-fmk or the broad spectrum inhibitor zVAD-fmk (both as fluoromethyl ketone derivatives). The cells were then treated with butyrate and 24 h cell lysates analyzed by western blotting. There was a consistent decrease in p15 with zDEVD-fmk and zVAD-fmk (Figure 5AGo). Suppression of p15 was more pronounced with zVAD-fmk and the band completely disappeared at a zVAD-fmk concentration of 100 µM. zDEVD-fmk gave only partial inhibition at concentrations up to 100 µM (the highest concentration tested). Cells treated with either zDEVD-fmk alone, zVAD-fmk alone or control cells untreated with butyrate showed no cleavage product (Figure 5AGo). Since DEVD-caspase activity in the cells was >90% suppressed at concentrations of >=0.6 µM for zDEVD-fmk (open circles in Figure 5BGo) and >=5 µM for zVAD-fmk (filled circles in Figure 5BGo), where inhibition of p21 cleavage was only partial, p21 may also be cleaved by other proteases.



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  		Fig. 5. Effect of caspase peptide inhibitors on p21 cleavage in butyrate-treated LIM 1215 cells. (A) Effect of inhibitors on the p15 band. Cells were treated with 4 mM butyrate for 24 h in the presence of the indicated concentrations of zDEVD-fmk or zVAD-fmk before assaying p21 cleavage by western blotting. Lane 1, butyrate-treated cells alone; lane 2, butyrate-treated cells + 50 µM zDEVD-fmk; lane 3, butyrate-treated cells + 100 µM zDEVD-fmk; lane 4, butyrate-treated cells + 50 µM zVAD-fmk; lane 5, butyrate-treated cells + 100 µM zVAD-fmk; lane 6, zDEVD-fmk alone; lane 7, zVAD-fmk alone; lane 8, control untreated cells. The figure shows partial suppression of p21 cleavage by zDEVD-fmk and complete suppression by 100 µM zVAD-fmk. (B) Effect of inhibitors on DEVD-caspase activity. {circ}, zDEVD-fmk; •, zVAD-fmk. The figure shows almost complete suppression of cellular caspase activity by both inhibitors at much lower concentrations than required to suppress p21 cleavage. These results are representative of three independent experiments.

 
Subsequent analysis of the p21 amino acid sequence revealed a likely caspase cleavage motif DHVD between amino acids 109 and 112 (Figure 6Go). Cleavage after D112 would yield fragments with predicted Mr of 12.4 and 6.0 kDa. The larger of these would be consistent with the size of the p15 fragment, since whole p21 has a calculated Mr of 18.4 kDa but migrates on SDS–PAGE with an apparent Mr of 21 kDa.



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  		Fig. 6. Structure of p21 showing the position of the caspase cleavage motif. The large rectangle depicts the 164 amino acid p21 protein with the N-terminal CDK-binding site (filled rectangle) and the C-terminal PCNA-binding site (hatched rectangle). The potential caspase cleavage motif DHVD occurs between these sites at positions 109–112. The arrow shows the proposed site of cleavage.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The aim of these studies was to explore further the mechanism by which butyrate regulates apoptosis and cell cycle arrest of colorectal cancer cells in vitro. Previously, we have demonstrated that butyrate induces apoptosis by a novel pathway initiated by inhibition of histone deacetylase and resulting in expression of a putative cell death gene which acts synergistically with the Bcl-2-regulated mitochondrial/cytochrome c-mediated pathway (9). These two pathways converge at a point upstream of caspase-3 protease, a major effector of apoptosis. We now show that induction of p21, and its cleavage, parallel in time these effects and may be critical regulatory events.

The induction of p21 by butyrate in LIM 1215 cells confirms studies in other CRC cell lines (13,19) and in other types of cell (20,21). LIM 1215 cells have wild-type p53 (25) and are likely to exhibit p53-dependent up-regulation of p21 in response to some stimuli (e.g. DNA-damaging agents). Further induction of p21 by butyrate may be a direct effect mediated by a butyrate-responsive element in the promoter region of the p21 gene (13). We now show that appearance of p21 immediately precedes the appearance of cytosolic DEVD-caspase activity and onset of DNA fragmentation. p21 was induced during an interval of 3–10 h, previously defined by our cycloheximide inhibition studies for expression of the essential pro-apoptotic protein (9). This raises the question of whether p21 is this protein or whether it is increased as a cellular protective mechanism against apoptosis. It seems unlikely that the butyrate-inducible pro-apoptotic protein is p21 since several studies have shown that p21 is anti-apoptotic. For example, antisense oligonucleotides which block p21 expression facilitate, rather than suppress, apoptosis in neuroblastoma cells (23). The observed dephosphorylation of hyper-phosphorylated Rb, beginning between 6 and 12 h after addition of butyrate to LIM 1215 cells, is in agreement with arrest of these cells in G1 (26). It is possible that by maintaining the cells in G1, p21 prevents critical events leading to caspase activation.

It is not yet known why some CRC cells differentiate in response to butyrate (3) while others die (7–9,27). The differentiation pathway is most prominent at 1 mM butyrate (3) while apoptosis is favored at 4 mM (9). However, even at 4 mM butyrate, a significant proportion of cells remained viable. A small proportion of these were killed by a second application of butyrate but no further cell loss occurred with a third cycle of butyrate. Since resistance to butyrate was lost following withdrawal of butyrate for 2 days, a butyrate-inducible factor may mediate the resistance to apoptosis. The increase in p21 expression in butyrate-resistant cells and its decrease in the absence of butyrate during the withdrawal phase implicates p21 as that factor.

The detection of an apparent fragment of p21 (designated p15) at later time points in butyrate-treated LIM 1215 cells was a surprising finding. Recently, the cleavage of p21 during apoptosis in response to various stimuli has been reported. These include cleavage of p21 in tumor necrosis factor-induced apoptosis of human cervical carcinoma cells (28), in growth factor-deprived human endothelial cells (29) and in response to DNA damage by {gamma}-irradiation and DNA-damaging agents (30,31). In support of the hypothesis that the p15 fragment seen here after butyrate treatment is a caspase cleavage product of p21: (i) p15 began to appear soon after DEVD-caspase was activated; (ii) it was greatly enriched in the floating apoptotic cell population, but not detected in butyrate-resistant LIM 1215 cells; (iii) its appearance was suppressed by peptide caspase inhibitors. That cleavage of p21 is not simply reflecting wide scale proteolytic degradation is suggested by the presence of a unique caspase-specific motif DHVD in p21 at a position that would correspond to one of the fragments having a molecular weight of ~15 kDa. Levkau et al. (29) showed p21 to be an apoptotic substrate which is cleaved at residue L112 and confirmation of this cleavage site was performed by site-directed mutagenesis. The latter study also showed cleavage of p27Kip1 in growth factor-deprived endothelial cells, but this was not observed in butyrate-treated LIM 1215 cells, suggesting that it may not be essential for apoptosis. The cleavage of p21 by caspases mimics cleavage by these proteases of certain other signal transduction proteins (32).

Our data have raised some anomalies regarding the relationship between caspases and cleavage of p21. Firstly, it is of interest to note that when caspase activity was restored in butyrate-resistant cells, following the withdrawal phase (Figure 3Go, lane Ad1'), there was only a weak band corresponding to the p15 fragment. We do not fully understand this, however, it may reflect differential subcellular localizations of both caspase and p21. Alternatively, the p15 fragment may be prone to further proteolytic degradation and therefore no longer detectable by western blotting. We are currently investigating these possibilities further. Secondly, there was only partial suppression of cleavage of p21 by zDEVD-fmk in butyrate-treated LIM 1215 cells, despite complete suppression of DEVD-caspase activity. This may indicate that other caspases and/or other proteases are involved in cleavage. It should be noted that p21 may also be cleaved via a proteasome-dependent mechanism (33).

Disruption of cell cycle regulatory checkpoints may be essential before suicide mechanisms can be turned on. In the absence of p21, DNA-damaged colon cancer cells undergo repeated S phases, without intervening mitoses, before dying by apoptosis (34). However, the rapidity of butyrate-induced apoptosis excludes this mechanism for killing of LIM 1215 cells by this agent. We have shown that cleavage of p21 in CRC cells precedes apoptotic detachment of cells from the substratum. This event might be important to apoptosis in either of two ways. Apoptosis may be allowed to proceed as a result of inactivation of p21 following cleavage, thereby disrupting its anti-apoptotic action, or, alternatively, the cleavage products themselves might actively promote apoptosis. Relevant to this, Prabhu et al. (35) showed that overexpression of a p21 truncation mutant, derived by introduction of a premature stop codon, in cancer cells induced apoptosis of the cells, whereas wild-type p21 only induced growth arrest. Coincidentally, the truncation mutant used in those studies terminated at L113, one amino acid beyond the putative DHVD-caspase cleavage site. Therefore, induction of apoptosis by overexpression of the caspase-derived fragment seems the more likely mechanism.

p21 is a polypeptide of 164 amino acids with a CDK-binding site in the N-terminal portion and a PCNA-binding site in the C-terminal portion (18). The significance of PCNA binding is unclear. Recently, Lu et al. (36) showed that mutant p21, lacking CDK-inhibitory activity but still retaining PCNA binding, no longer protected a CRC cell line from induction of apoptosis by X-rays or adriamycin, implying that inhibition of CDK is critical to p21-mediated suppression of apoptosis. In another study, mutants lacking PCNA binding were unable to arrest growth, although apoptosis was not investigated (37). Since the putative DHVD cleavage motif lies between the CDK–cyclin- and PCNA-binding domains (Figure 6Go), cleavage may disrupt the function and/or subcellular localization of p21. One possibility is that p15, which still retains the CDK–cyclin-binding domain, competes with the remaining p21 for CDK but is unable to inhibit it because PCNA is no longer part of the complex. Alternatively, p15 may fail to localize in the nucleus because it lacks PCNA binding.

Therefore, expression of p21 and its cleavage are likely to have important implications, not only for colorectal tumorigenesis but also for mechanisms of regulation of cell turnover in normal intestinal epithelium. Expression of p21WAF1/CIP1 marks the transition from proliferation to terminal differentiation in intestinal epithelium (22). Cleavage of p21 may, in turn, mark the transition from terminal differentiation to apoptosis at the luminal ends of the crypts. The importance of caspase-mediated turnover of p21 versus that of transcriptional regulation now needs to be defined. It is possible that cleavage of p21 is a novel regulatory mechanism that facilitates apoptotic death of colon cancer cells by butyrate and perhaps other stimuli.


   Acknowledgments
 
We would like to acknowledge Dr R.Whitehead for supply of colorectal cancer cell line LIM 1215. We also thank Ms Liza Raggatt for assistance with FACS analysis. F.C. was supported by an overseas program research scholarship. This work was, in part, supported by grants from the Anti-Cancer Foundation of South Australia, the Australian Research Council and the University of Adelaide.


   Notes
 
3 To whom correspondence should be addressed Email: pzalewski{at}medicine.adelaide.edu.au Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 12, 1999; revised September 16, 1999; accepted September 21, 1999.


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