School of Biological Sciences, University of Sussex, Brighton, East Sussex, BN1 9QG, UK1
Author for correspondence: Alison Sinclair. Fax +44 1273 678433. e-mail A.J.Sinclair{at}sussex.ac.uk
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Abstract |
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Introduction |
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The high abundance of p27KIP1 protein observed in quiescent primary B-cells is a feature common to many non-proliferating cells in vivo (Fero et al., 1996 ; Fredersdorf et al., 1997
; Kiyokawa et al., 1996
; Nakayama et al., 1996
). In addition, in many in vitro cell culture systems the level of p27KIP1 protein is inversely regulated with the proliferation state (Bartek, 1996
; Eblen et al., 1995
; Firpo et al., 1994
; Hengst et al., 1994
; Herwig, 1997
; Kwon et al., 1996
; Nourse et al., 1994
; Polyak et al., 1994
; Ravitz et al., 1995
; Reynisdottir et al., 1995
; Slingerland, 1994
).
Regulation of p27KIP1 is achieved through several complementary routes involving transcriptional control (Kawamata et al., 1998 ; Kwon et al., 1996
, 1997
), altered translation (Agrawal et al., 1996
; Hengst & Reed, 1996
; Millard et al., 1997
), sequestration (Barnouin et al., 1999
; Polyak et al., 1994
; Poon et al., 1995
; Reynisdottir et al., 1995
; Sherr & Roberts, 1999
; Soos et al., 1996
; Vlach et al., 1996
) and ubiquitin-dependent degradation by the proteasome (Pagano et al., 1995
).
A further layer of regulation of p27KIP1 expression involving cleavage by caspases has recently been described in various cell models including endothelial cells undergoing growth factor deprivation-induced apoptosis (Levkau et al., 1998 ), apoptosis induced by a variety of agents in myeloid leukaemia cells (Eymin et al., 1999
), growth-arrested hybridoma and myeloma cell lines (Loubat et al., 1999
) and proliferating BJAB B-lymphoblastoid cells (Frost et al., 2001
). In addition, we have shown that p27KIP1 is down-regulated by both caspase-dependent and -independent pathways in lymphoid cells undergoing apoptosis (Frost & Sinclair, 2000
). p27KIP1 contains two potential caspase recognition sites, DPSD139 and ESQD108 (Eymin et al., 1999
). Cleavage at these sites results in C-terminally truncated p27KIP1 proteins of 23 kDa and 15 kDa respectively (Eymin et al., 1999
); the 23 kDa product (p23) is more frequently observed (Eymin et al., 1999
; Frost et al., 2001
; Levkau et al., 1998
; Loubat et al., 1999
). At least two different, as yet unidentified, caspases with differing sensitivity to z-VAD-fmk have the potential to generate p23 (Eymin et al., 1999
; Frost et al., 2001
; Levkau et al., 1998
; Loubat et al., 1999
).
In this report we extend the role of caspase or caspase-like enzymes to the regulation of p27KIP1 protein abundance in cells immortalized by EBV. We confirm that p27KIP1 is significantly down-regulated following immortalization of primary B-lymphocytes by EBV and that the resulting LCLs consistently contain a low basal level of p27KIP1 protein; this is not present in sufficient abundance to abrogate cdk2 activity. Comparison with uninfected primary B-lymphocytes demonstrates that the regulation is achieved at a post-translational level. Ubiquitin-mediated degradation may contribute to the regulation but a more significant role is played by caspase cleavage. A caspase activity that recognizes DPSD139 of p27KIP1 can be readily detected in EBV-immortalized LCLs, in the absence of apoptosis, and this activity correlates with the presence of a 23 kDa truncated form of p27KIP1 in LCLs. Interestingly, the caspase activity is similar to one previously characterized in an EBV-negative B-lymphoma-derived cell line (BJAB) (Frost et al., 2001 ), which indicates that cleavage by a proliferation-associated caspase may represent a general mechanism by which p27KIP1 expression is controlled in lymphoid cell lines.
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Methods |
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Western blotting.
Total protein extracts were prepared from exponentially growing cell lines and from quiescent primary B-cells as described previously (Cannell et al., 1996 ). Unless otherwise indicated, the protein concentrations were normalized after quantifying the absorption at 280 nm and 100 µg of each extract was fractionated on an SDSpolyacrylamide gel, transferred to PVDF membrane (Immobilon-P) and processed as previously described (Cannell et al., 1996
). An additional gel was used to assess the equality of the protein concentrations by Coomassie blue staining. The p27KIP1 polyclonal antibodies C19 and N20, which are specific for the C and the N termini of p27KIP1 respectively, were purchased from Santa Cruz. The p53 monoclonal antibody DO1 was as described by Vojtesek (1992)
. Signals from the primary antibodies were amplified using species-specific antisera conjugated to either horseradish peroxidase (Amersham) or alkaline phosphatase (Sigma) and detected using either ECL or ECF (Amersham). Signals detected by ECL were quantified after autoradiography using Imagemaster software (Pharmacia). Signals detected by ECF were quantified directly using the Storm phosphorimager and ImageQuantNT software (Molecular Dynamics).
cdk2 inhibition.
Roscovitine (Calbiochem) was solubilized in DMSO. Exponentially growing cells were cultured in the presence of roscovitine (150 µM) or DMSO for 8 h. The rate of DNA synthesis was determined by measurement of thymidine incorporation during a terminal 4 h pulse in medium containing [3H]thymidine (5·0 µCi/ml; 185 kBq/ml). The cells were harvested onto paper using a semi-automatic cell harvester (Skatron) and the amount of thymidine incorporated into DNA was determined by liquid scintillation counting. Assays were carried out in triplicate and standard deviations are shown.
Immunoprecipitation.
NP40 extracts were prepared from exponentially growing cells as previously described (Cannell et al., 1996 , 1998
). Briefly, cells were washed in PBS and lysed in NP40 lysis buffer (50 mM HEPES; 1%, v/v, NP40; 0·1%, v/v, Tween 20; 150 mM NaCl; 1 mM EDTA; 2·5 mM EGTA; 1 mM NaF; 10%, v/v, glycerol; pH 8·0). After removal of debris by centrifugation the extract was pre-cleared with protein ASepharose. Cleared extract derived from 4·3x107 cells was used in each immunoprecipitation with either 4 µg of non-specific rabbit immunoglobulin, anti-p27KIP1 (Santa Cruz, C19) or anti-cdk2 (Santa Cruz, M2). Mock immunoprecipitations performed in the absence of extract were performed for each antibody. Antibodyprotein complexes were collected with protein ASepharose, and washed extensively with NP40 lysis buffer. After a final wash with 50 mM TrisHCl, pH 7·5, proteins were eluted by boiling in SDS sample buffer lacking reducing agents (4%, w/v, SDS; 20%, v/v, glycerol; 100 µg/ml bromophenol blue; 0·12 M TrisHCl, pH 6·8). The amount of p27KIP1 and cdk2 in the immunoprecipitated proteins derived from 1·6x107 cells was detected by Western blotting using either anti-p27KIP1 (Santa Cruz, C19) or anti-cdk2 (Santa Cruz, M2) as the primary antibody, followed by ECL.
Protein half-life determination.
Cells were incubated at a density of 1x106 cells/ml in fresh medium for 1 h. Anisomycin was added to a final concentration of 100 µM. Cells were harvested over an 8 h period and total protein extracts were prepared, fractionated on an SDSpolyacrylamide gel and analysed by Western blotting.
Proteasome inhibition.
Cells were resuspended in fresh medium at a density of 2·5x106 cells/ml and treated with proteasome inhibitors for a total of 320 min. The proteasome inhibitors lactacystin and carbobenzoxy-L-leucyl-leucyl-leucinal (MG-132) were obtained from Calbiochem and used at final concentrations of 100 µM and 10 µg/ml respectively. Calpain inhibitor II, L-acetyl-leucyl-leucyl-methionyl (LLM), was obtained from Sigma and was used at a final concentration of 10 µg/ml.
Apoptosis assays.
The Roche Annexin-V-FLUOS staining kit was used to detect cell surface expression of phosphatidylserine as an early marker of apoptosis. The amount of cell surface-associated annexin Vfluorescein in a population of live cells was quantified using a FACS Calibur (Becton Dickinson) according to instructions supplied with the staining kit. In parallel, exponentially growing cells were treated with an agonistic Fas antibody as previously described (Frost & Sinclair, 2000 ) to generate a positive control for apoptosis. In addition, exponentially growing and Fas-ligated cells were deposited onto glass slides, fixed with paraformaldehyde (4%, v/v, in PBS), and stained with the DNA-specific dye Hoechst 33342 (0·1 mg/ml). The cells were inspected for nuclear degeneration by fluorescence microscopy and photographed.
Caspase assays.
Extracts were prepared and fluorogenic caspase assays were carried out as previously described (Frost et al., 2001 ). Briefly, cells were washed with PBS and lysed in buffer containing 130 mM NaCl, 1% (v/v) Triton X-100, 10 mM NaPPi, 10 mM TrisHCl, 1 mM EDTA, 1 mM PMSF, 0·25 µg/ml Pepstatin A, 10 mM NaH2PO4/Na2HPO4, pH 7·5. Debris was removed by centrifugation and the cell extract collected. The ability of the resultant cell extract to cleave the three tetrapeptideAMC substrates Ac-IETD-AMC, Ac-DPSD-AMC and Ac-ESQD-AMC was monitored as previously described (Frost et al., 2001
). Briefly, 50 µl of the resultant extract was mixed with 200 µl reaction buffer (10%, v/v, glycerol; 2 mM DTT; 1 mM EDTA; 1 mM PMSF; 0·25 µg/ml Pepstatin A; 200 mM HEPES, pH 7·5; 25 µg/ml tetrapeptideAMC substrate) in a 96-well plate. The caspase inhibitors Ac-IETD-CHO (2 µg/ml), iodoacetamide (50 mM) and z-VAD-fmk (3 µM) were also added where indicated. Reactions were allowed to proceed for 2 h at 37 °C before the AMC liberated from the tetrapeptideAMC substrates was measured using a SpectraMax Gemini spectrofluorometer (Molecular Devices); excitation wavelength 380 nm, emission wavelength 440 nm. Background measurements generated from the reaction completed in the absence of substrate and extract were assessed and subtracted from experimental values.
Caspase inhibition in cultured cells.
z-VAD-fmk (Calbiochem) and z-IETD-fmk (BioVision) were solubilized in DMSO and added to exponentially growing IB4 cells at the concentrations indicated. Alternatively, cultures were allowed to saturate over a 5 day period and then stimulated to re-enter the cell cycle by dilution to 2x105 cells/ml in the presence of either 2 µM z-IETD-fmk or DMSO.
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Results |
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cdk2 is required for the continued proliferation of EBV-immortalized B-cell lines
We sought to question whether the activity of cdk2 complexes is required for the continued proliferation of LCLs. A specific inhibitor of cyclin-dependent kinases, roscovitine (Meijer, 1996 ), was applied to an EBV-immortalized cell line, IB4. Inhibition of DNA synthesis was clearly observed 8 h after the addition of 25 µM and 50 µM roscovitine (Fig. 1C
). Roscovitine has been shown to selectively inhibit cdc2, cdk2 and cdk5 in vitro (Meijer, 1997
). Since the kinase activity of cdk5 is restricted to neuronal cells (Tsai, 1993
), we conclude that the experiment demonstrates a dependence of LCLs on cdc2 and/or cdk2. The involvement of these kinases can be distinguished by analysing the cell cycle profile of the arrested cells; inhibition of cdk2 arrests cells in G1 with a 2n DNA content whereas inhibition of cdc2 arrests cells in G2 with a 4n DNA content. Since 60·2% of IB4 cells treated with roscovitine are arrested in G1 (data not shown), a role for cdk2 for the continual proliferation of the cells is clear. This underlines the relevance of the EBV-associated down-regulation of p27KIP1 to the continual proliferation of LCLs.
The half-life of p27KIP1 is reduced following immortalization of primary B-lymphocytes with EBV
Bearing in mind the proposed relevance of p27KIP1 down-regulation to the proliferation of EBV-immortalized cells, we were interested to elucidate the mechanism(s) by which the down-regulation is achieved. This was first approached by examining the level of mRNA encoding p27KIP1 in quiescent primary B-cells and LCLs (IB4 and LCL#3). No significant differences in the amount of p27KIP1 mRNA were detected (data not shown), suggesting that the mechanism responsible for reduced expression of p27KIP1 in the LCLs was post-transcriptional.
The half-life of p27KIP1 protein was assessed in quiescent primary B-cells and LCLs (IB4). Novel protein synthesis was halted by the addition of anisomycin and the steady state level of the pre-existing p27KIP1 protein within the cell was determined over the following 8 h. During this time period the treated cells remained viable, as judged by trypan blue exclusion assays. p27KIP1 protein appears to be very stable in quiescent primary B-cells and there was little diminution in the level observed over the 8 h of the experiments (Fig. 2, top). In contrast p27KIP1 protein in IB4 cells was turned over with a half-life of 3·2±1·5 h (Fig. 2
, bottom) and a similar half-life of 3·0±0·7 h was obtained for p27KIP1 protein in LCL#3 cells (data not shown). The half-life of p27KIP1 protein determined in LCLs is similar to that observed in other types of proliferating cells (Hengst & Reed, 1996
).
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The ubiquitin-mediated proteasomal degradation pathway has been implicated in the regulation of p27KIP1 protein in a number of cell systems (Alessandrini et al., 1997 ; Pagano et al., 1995
); ubiquitin-conjugated forms of p27KIP1 can be found within cells and the addition of cell-permeable proteasome inhibitors results in an accumulation of p27KIP1 protein within 90 min (Pagano et al., 1995
). In order to question whether the degradation of p27KIP1 that we observe in LCLs is mediated by the proteasome, we asked whether inhibiting proteasome function influences the steady state levels of p27KIP1 protein in IB4 cells. Neither the proteasome-specific inhibitor lactacystin nor the proteasome and calpain inhibitor MG-132 (LLL) induced an increase in the level of p27KIP1 protein in LCLs during the period of the experiment (Fig. 3
), although the level of another protein that is known to be regulated by this pathway, p53, was clearly increased by three- to eightfold during this time-frame. In addition, the calpain II inhibitor LLM failed to increase the level of p27KIP1 protein in LCLs, although it was able to elevate p53 levels within the same cells, as has been described previously (Kubbutat, 1997
) (Fig. 3
). The use of proteasome inhibitors in many transformed cells, including LCLs is, complicated by their tendency to promote apoptosis in these cells (Drexler, 1997
; M. Hayes & A. J. Sinclair, unpublished data). We previously demonstrated that p27KIP1 is down-regulated during apoptosis of lymphoid cells, including LCLs (Frost & Sinclair, 2000
). It is therefore possible that proteasome inhibitors may inhibit ubiquitin-mediated degradation of p27KIP1, whilst simultaneously promoting apoptosis, resulting in the down-regulation of p27KIP1. To address this, we closely monitored the viability of the cells throughout the incubation period. From the data presented here it appears that the proteasome degradation pathway does not play a major role in the regulation of p27KIP1 protein in LCLs.
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Western blotting of extracts from proliferating IB4 cells with an antibody directed at the N terminus of p27KIP1 demonstrates that IB4 cells, like BJAB cells, contain a cleaved form of p27KIP1 (Fig. 4A). Note that this species would not have been detected in the previous figures. The migration of the protein was consistent with truncation at D139 (data not shown). The presence of a putative caspase cleavage product in proliferating IB4 cells led us to examine whether a significant proportion of the cells were undergoing apoptosis. As a positive control for apoptosis IB4 cells were treated with an agonistic Fas antibody for 2 h and both early and late signs of apoptosis were monitored. Fig. 4(B)
shows a dramatically different profile of annexin V-binding in populations of proliferating or Fas-ligated IB4 cells. Whereas the majority of the proliferating cell population (85%) shows low affinity for annexin V, and hence does not express significant amounts of cell surface phosphatidylserine, an early marker for apoptosis, the Fas-ligated cells show a large rightwards shift in annexin V-binding, indicative of the onset of apoptosis in the majority (80%) of the population. Staining with the DNA-specific dye Hoechst 33342 confirmed these differences (Fig. 4C
, D
). As expected, many cells in the Fas-ligated population had entered the late stages of apoptosis, with hallmark popcorn-like nuclear morphology (Fig. 4D
). However, we failed to detect any late apoptotic cells in the proliferating population (Fig. 4C
) despite the presence of a small proportion of annexin V-binding cells in the population (Fig. 4B
). Thus, similar to proliferating EBV-negative BJAB cells, EBV-immortalized IB4 cells contain a putative caspase cleavage product of p27KIP1, and this appears to arise independently of apoptosis.
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Thus, EBV-immortalized B-cells contain a caspase or caspase-like activity that is able to cleave the DPSD139 caspase recognition site present in p27KIP1. The presence of a C-terminally truncated form of p27KIP1 in LCLs whose size is consistent with cleavage at the site supports the notion that caspase cleavage plays a significant role in maintaining the low basal level of p27KIP1 abundance in EBV-immortalized cells.
Selective inhibition of caspase(s) in EBV-immortalized cells results in a dramatic inhibition of proliferation
The importance of caspase activity(s) for the continued proliferation of EBV-immortalized cells was investigated using the cell-permeable caspase inhibitors z-VAD-fmk and z-IETD-fmk. Exponentially growing IB4 cells were treated with increasing concentrations of the two inhibitors and DNA synthesis was measured 24 h later. As shown in Fig. 6, the proliferation of IB4 cells was largely unaffected by concentrations of z-VAD-fmk up to 75 µM. This eliminates any z-VAD-fmk-sensitive caspase (including caspase-8) from playing a major role in the proliferation of IB4 cells. In contrast, treatment of IB4 cells with comparatively low concentrations of z-IETD-fmk (110 µM) produced a dramatic inhibition of DNA synthesis (Fig. 6
). This resembles the inhibition of DNA synthesis previously described in exponentially growing BJAB cells treated with z-IETD-fmk (Frost et al., 2001
). These observations implicate a caspase with sensitivity to z-IETD-fmk but resistance to z-VAD-fmk in regulating the proliferation of IB4 cells. As this inhibitor profile matches that of the DPSD-directed caspase activity detected in vitro, we investigated whether the expression of p27KIP1 in IB4 cells was affected by treatment with z-IETD-fmk.
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Discussion |
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We and others have previously demonstrated that the retinoblastoma protein phosphorylation pathway is activated following infection of primary B-cells with EBV (Allday et al., 1995 ; Cannell et al., 1996
; Hollyoake et al., 1995
; Kempkes, 1995
; Pokrovskaja et al., 1999
; Sinclair et al., 1994
, 1998
; Spender et al., 1999
, Szekely et al., 1995
) and we suggest that this pathway is required to facilitate the transition of infected cells through the restriction point. By analogy with other cell systems, the key kinases that direct the phosphorylation of pRb are (i) cyclin Dcdk4 and/or cyclin Dcdk6 and (ii) cyclin Ecdk2. The expression of all of these components is induced following the immortalization of B-cells by EBV (Cannell et al., 1996
; Hollyoake et al., 1995
; Kempkes, 1995
; Sinclair et al., 1994
, 1998
; Szekely et al., 1995
). In this report, we show that inhibition of cdk2 kinase activity arrests LCL cells, implying that cdk2 activity and the phosphorylation of the retinoblastoma protein are required for the continued proliferation of LCLs.
We further questioned how the cdk2 activity is regulated following EBV-mediated immortalization. In part this is achieved by de novo synthesis of both cdk2 and cyclin E (Cannell et al., 1996 ; Hollyoake et al., 1995
; Kempkes, 1995
; Sinclair et al., 1994
). However, the precursor quiescent primary B-cells contain a high level of the cyclin Ecdk2 inhibitor p27KIP1 (Cannell et al., 1996
; Spender et al., 1999
), which displays inhibitory activity against cdk2 in vitro.
The importance of p27KIP1 protein to the regulation of cell proliferation is underscored by the myriad of molecular mechanisms that have evolved to regulate the level of p27KIP1 protein within cells (see Introduction). This description of caspase-mediated regulation of p27KIP1 in EBV-immortalized B-cells, coupled with similar observations in the BJAB B-lymphoma cell line (Frost et al., 2001 ), suggests caspase cleavage may represent a widespread mechanism for regulating p27KIP1 expression in lymphoid cells (see below). The potential for further regulation of p27KIP1 by caspases during apoptosis remains open. It is now well established that caspases have roles in processes other than apoptosis. Caspase-1, -4, -5 and -13, which share a preference for bulky hydrophobic residues at position 4 of the tetrapeptide caspase recognition sequence (defined as group I caspases), have a role in cytokine processing. Indeed, this substrate specificity is at odds with a role for these caspases in apoptosis, as none of the polypeptides so far identified that are cleaved during apoptosis contain hydrophobic residues at position 4 (Nicholson, 1999
). Even group II caspases, which have a substrate specificity consistent with many apoptosis-related substrates (DEXD), can be found activated in the absence of apoptosis, suggesting roles in other cellular processes (Alam et al., 1999
; Miossec et al., 1997
). The substrate specificity of p27KIP1-directed caspases (DPSD) conforms most closely to a type II caspase recognition motif. However, the presence of proline at position 2 appears to preclude many known caspases (Thornberry et al., 1997
). The insensitivity of the DPSD-directed caspase we have described herein and in proliferating BJAB cells (Frost et al., 2001
) to z-VAD-fmk also distinguishes the caspase from many previously described (Garcia-Calvo et al., 1998
). The presence of an apparently identical DPSD-directed caspase activity in both an EBV-negative lymphoma-derived cell line (Frost et al., 2001
) and in an EBV-transformed B-cell line indicates that EBV activates a normal cellular enzyme to negate the functions of p27KIP1 during EBV-mediated immortalization. At present it is unclear whether the caspase activity is restricted to transformed lymphoid cells (a similar activity is also present in proliferating Jurkat T-cells; Frost et al., 2001
) or whether the caspase is active in proliferating cells of more diverse lineages; this is currently under investigation in our laboratory.
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Acknowledgments |
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Received 13 June 2001;
accepted 8 August 2001.