peptides 28 (2007) 731–740
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A sychnological cell penetrating peptide mimic of p21WAF1/CIP1 is pro-apoptogenic Rachael D. Baker, John Howl, Iain D. Nicholl * Research Institute in Healthcare Science, School of Applied Sciences, University of Wolverhampton, Wolverhampton WV1 1SB, United Kingdom
article info
abstract
Article history:
Targeting chemotherapeutic agents directly to sites of DNA replication and repair within
Received 2 November 2006
cancerous cells is problematic. This study attempts to address the issue of nuclear delivery
Received in revised form
of biologically active peptides with the potential to disrupt cancer cell growth. Herein, the
5 December 2006
protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat48–60), is
Accepted 5 December 2006
used to deliver a cytotoxic peptide mimic of the cyclin-dependent kinase inhibitor, p21WAF1/
Published on line 27 December 2006
CIP1
into the nucleus. This construct, which we designate as Tat48–60-P10, contains the PCNA
interacting protein (PIP) box. We demonstrate the utility of Tat48–60 for peptide delivery to the Keywords:
nucleus and show that Tat48–60-P10 induces apoptosis specific to the inclusion of the wild
Tat
type PIP box containing sequence. Colocalization of Tat48–60-P10 with nuclear PCNA was
Proliferating cell nuclear antigen
observed by immunofluorescence analysis, supporting the hypothesis that cytotoxicity is
(PCNA)
potentially related to disruption of nuclear PCNA function. The U251 and U373 glioma cell
Apoptosis
lines exhibited particular sensitivity to the construct. # 2007 Elsevier Inc. All rights reserved.
Cell penetrating peptide p21WAF1/CIP1
1.
Introduction
Malignant gliomas are the most common primary brain tumor type and despite aggressive chemotherapy the average survival rate for a patient can be less than 1 year [10,24]. A major difficulty with the use of alkylating agents for the treatment of astrocytomas is the chemoresistance that these neoplasms demonstrate [24]. As disrupting proliferating cell nuclear antigen (PCNA) function can sensitize cells to alkylating agents [14], we hypothesized that the disruption of PCNA using a peptide mimetic of the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, may be cytotoxic and provide a novel approach to sensitize cells to chemotherapeutic agents. PCNA is a 36 kD polypeptide critically involved in DNA replication and repair [5,22,35], and regulates numerous protein–protein interactions by acting as molecular scaffold-
ing for the proteins involved [17,25]. Originally identified as cyclin, this cell cycle regulated protein was discovered to be necessary for the elongation stage of simian virus 40 DNA replication and acts as a processivity factor of polymerase d in human DNA replication [3,28]. Numerous PCNA–protein interactions are mediated via a PCNA interacting protein (PIP)-box consensus sequence, (QXX(I/L/M)XX(F/H/D)(F/Y), where X is any amino acid, found in cognate partners of PCNA [34]. For example, DNA polymerase d, MSH2/MSH6 (human DNA mismatch repair complex) and also the cyclindependent kinase (CDK) inhibitor – the p21WAF1/CIP1 tumor suppressor protein – are posited to interact with PCNA via their respective PIP-box. In vivo, DNA damage stimulates p53 activation resulting in p21WAF1/CIP1 expression. Upregulated p21WAF1/CIP1 inhibits cyclin-dependent kinases and concomitant DNA replication
* Corresponding author. Tel.: +44 1902 321134; fax: +44 1902 322714. E-mail address:
[email protected] (I.D. Nicholl). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.12.013
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peptides 28 (2007) 731–740
[12,19,22]. Additionally, the carboxy-terminal domain of p21WAF1/CIP1 can bind the interdomain connector loop (ICL) of PCNA thus competing with the DNA polymerase d-PCNA interaction blocking PCNA-dependent DNA replication [6,17]. As it has been suggested that small analogs mimicking p21WAF1/CIP1 may disrupt PCNA function [12], herein, a sychnologically organized peptide [27] was constructed based on the p21WAF1/CIP1 PIP-box, designated ‘P10’ [1,20,22,35], with cell and nuclear penetration achieved by the Tat48–60 peptide, derived from the HIV-1 transactivator of transcription protein, Tat. The cell penetrant properties of Tat [9,11], and the capacity of Tat to carry molecular cargoes into cells is well established [8]. In this study, Tat48–60 was selected as it efficiently retains the penetrant properties of the full length HIV-1 Tat protein [33], is an effective vehicle for the delivery of biologically active peptide cargoes [16], retains a nuclear localization signal (NLS), has relatively low intrinsic toxicity [30], and was thus considered to be an ideal candidate to deliver the P10 peptide to the nucleus. Tat48–60-P10 cytotoxicity was assessed and compared to control peptides with alanine mutations in the conserved residues of the p21WAF1/CIP1 PIPbox and/or deletions of the basic Tat48–60 fragment, and the cellular localization of the Tat48–60 containing peptide constructs elucidated. Our data suggest that the peptide cargo reached the nucleus and, as far as we are aware, this is the first time that a sychnological cell penetrating peptide mimic of p21WAF1/CIP1 has been colocalized with its cognate partner, PCNA, and demonstrates the cytotoxicity of the peptide to a range of cancer cells in vitro, which we propose – based on colocalization and in vitro binding – is associated with the disruption of PCNA function, ultimately triggering apoptosis.
2.
Materials and methods
2.1.
Cell culture
U251 (glioblastoma, grade IV) was grown in Hams F10. SW480 (colorectal adenocarcinoma) were cultured in L-15 Leibovitz [18] medium supplemented with 1% L-Glutamine/Penicillin/ Streptomycin. U373 (astrocytoma, grade III) was grown in DMEM (PAA Laboratories) supplemented with 1% Penicillin/ Streptomycin. MCF7 (breast carcinoma) cells were cultured in MEM supplemented with 1% L-Glutamine/Penicillin/Streptomycin and 1% non-essential amino acids. All media was supplemented with 10% (v/v) fetal calf serum (PAA). All cells were cultured at 37 8C in a humidified atmosphere (including 5% CO2 for U373 and MCF7). All reagents were provided by Sigma Chemical Co., unless otherwise indicated. Cell lines were provided by European Collection of Cell Culture (ECACC) except U251 (Institute of Neurology, Queen Square, London).
were purified to homogeneity by HPLC (Genesis C18 column) and the predicted masses of all peptides used in this study (average M+ H+) were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Kratos Analytic Kompact Probe operated in a positive ion mode) (supplementary data available on request). Purified peptides were lyophilized, suspended in sterile phosphate buffered saline (PBS) at a concentration of 1 mM and stored at 80 8C until required. Nterminal sequencing (Edman degradation; performed by Alta Biosciences, University of Birmingham) additionally validated the Tat48–60 peptide sequence (data not shown).
2.3.
Cytotoxicity induced by the peptide constructs described was quantified by a 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, as previously described [21]. Briefly, cells were seeded in 96 well Sarstedt plates (104 cells/well) for 24 h prior to treatment. Following peptide exposure for 24 h, 0.5 mg/mL of the tetrazolium salt, MTT (Sigma) was added to each well for 3 h after which time formazan products were solubilized with DMSO (Sigma). The reduction of MTT to formazan was then measured at 540 nm using a microplate reader (Labsystems Multiskan MS). The toxicity of each peptide treatment was reported as a percentage of the untreated control.
2.4. The specific binding of Tat48–60-P10 to PCNA as demonstrated by immunoblotting BSA (negative control), Tat48–60-P10[Ala 15,18,21] and Tat48–60-P10 were dotblotted onto nitrocellulose membrane, all at an equimolar concentration of 1 mM and incubated with 33 mg/ mL of recombinant PCNA protein in PBS, U251 total cell extract in PBS and PBS alone for 48 h at 4 8C. The U251 cell lysate was prepared by collecting trypsinized cells in media and centrifugation for 2 min at 1000 rpm to obtain a cell pellet, which was washed twice with PBS. The cell pellet was resuspended in a mild lysis buffer (10 mM Tris–HCl, pH 8 (Sigma), 0.15 M NaCl (Sigma), 5 mM EDTA (Sigma) and 0.5% (v/ v) NP40 (BDH)) and vortexed briefly, before incubating on ice for 1 h. After centrifugation at 13,000 rpm for 15 min, the supernatant was collected and stored at 20 8C prior to use. Immunoblotting was performed under standard conditions using monoclonal anti-human PCNA (clone PC10, Sigma) primary antibody and goat anti-mouse HRP (Sigma) secondary antibody. Western blot analysis of U251 cell nuclear extract and recombinant PCNA and Coomassie staining of recombinant PCNA were used to confirm the presence of PCNA within U251 extracts and the purity of the recombinant PCNA.
2.5. 2.2.
Cytotoxicity of peptides in human carcinoma cell lines
Cellular localization of Tat48–60-P10
Peptide preparation
Solid phase peptide synthesis employed a standard N/-Fmoc (N-(9-fluorenyl)methoxycarbonyl) protection strategy on rink amide MBHA resin (NovaBiochem) with HCTU (AGTC Bioproducts Ltd.)/HoBt (Advanced ChemTech) activation. Synthesis was carried out on a 0.2 mmol scale with Kaiser tests used at each step to confirm coupling and/or deprotection [15]. Peptides
U251 cells were seeded at a concentration of 5 104 cells/well on glass coverslips and cultured for 24 h in the indicated media with 10% FCS. Cells were subsequently incubated with 50 mM Tat48–60-P10, Tat48–60-P10[Ala 15, 18, 21] or Tat48–60 for 24 h, washed with PBS and fixed with methanol/acetone. Fixed cells were then blocked with 3% (w/v) BSA with 0.2% (v/v) Tween in PBS and incubated with a 1/100 monoclonal anti-Tat antibody
peptides 28 (2007) 731–740
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directed against the Tat (49–58) peptide [33] (provided by Dr. Eric Vives, Institut de Ge´ne´tique Mole´culaire de Montpellier, France) for 1 h at room temperature. Coverslips were washed with 1% (w/v) BSA with 0.2% (v/v) Tween in PBS then incubated with 1/200 goat anti-mouse FITC secondary antibody (Sigma) for 1 h. After a final wash step, coverslips were mounted on slides with VectashieldTM containing DAPI (Vector Laboratories Inc.) and analyzed by immunofluorescence microscopy (Olympus BX61) with excitation/emission wavelengths set at 490/525 nm for FITC and 350/460 nm for DAPI. Colocalization studies were also carried out using the methodology described above with the following inclusions; 1/50 goat anti-PCNA (C20) (Santa Cruz Biotechnology Inc.) was used as a primary antibody in conjunction with a 1/100 dilution of the monoclonal anti-Tat antibody. 1/100 rabbit anti-goat IgG-R (Santa Cruz Biotechnology Inc.) and 1/200 goat anti-mouse FITC were utilized as secondary antibodies. Excitation/emission wavelengths were set at 570/620 nm for rhodamine (Olympus BX61).
Assay of cell death mechanism triggered by Tat48–60-
2.6. P10
A canonical feature of apoptosis, nuclear DNA fragmentation, was detected employing the TUNEL assay (In situ Cell Death Detection Kit, TMR Red, Roche Applied Science) according to the manufacturer’s instructions. Briefly, U251 cells were seeded at a concentration of 5 104 cells/well in 12 well plates (Nunc) with glass coverslips and cultured for 24 h in the indicated media with 10% (v/v) FCS. Cells were subsequently treated with 50 mM Tat48–60-P10 or Tat48–60-P10[Ala 15, 18, 21] for 24 h. Cells were washed with phosphate buffered saline (PBS) pH 7.4 prior to fixation with 4% (w/v) paraformaldehyde in PBS (Sigma) for 1 h at room temperature. Cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma) in 0.1% sodium citrate for 2 min at 4 8C. Incubation with TUNEL reaction mixture containing Tdt and TMR red-dUTP was carried out in the dark at 37 8C in a humidified atmosphere for 1 h. Coverslips were washed with PBS, mounted on slides with VectashieldTM containing DAPI and analyzed by fluorescence microscopy (Olympus BX61) with excitation/emission wavelengths set at 570/620 nm for TMR red and 350/460 nm for DAPI.
3.
Fig. 1 – (a) Cell viability of SW480 human adenocarcinoma cells when treated with the cell penetrant peptide, Tat48– 60 -P10, and control peptides, Tat48–60-P10[Ala 15,18,21], P10 and P10[Ala 15,18,21]. The concentration range of all peptides used was 0–80 mM for 24 h at 37 8C. Note increased cytotoxicity of Tat48–60-P10 compared to control peptides. Error bars indicate S.E.M. (n > 3). (b) Cell viability of U251 human glioblastoma cells when treated with the cell penetrant peptide, Tat48–60-P10, and control peptides, Tat48–60-P10[Ala 15,18,21], P10 and P10 [Ala 15,18,21]. The concentration range of all peptides used was 0–80 mM for 24 h at 37 8C. Note increased cytotoxicity of Tat48–60-P10 at 40 mM when compared to control peptides. Error bars indicate S.E.M. (n > 3).
Results
The peptides used in this study are shown in Table 1: Tat48–60 is the minimal protein transduction domain of the HIV-1 Tat protein; P10 is the PCNA interacting region or ‘PIP’ box of
Table 1 Designation
Peptide sequence
48–60
Tat Tat48–60-P10 Tat48–60-P10[Ala P10 P10[Ala
15,18,21]
p21WAF1/CIP1 (amino acids 143–160 of p21); Tat48–60-P10 is a tandem chimeric peptide composed of Tat48–60 and p21WAF1/ CIP1(143-160) ; Tat48–60-P10[Ala 15,18,21] is a tandem chimeric peptide with the key residues required for PCNA binding substituted with alanine. Neither P10 nor P10[Ala 15,18,21] contain the Tat48– 60 fragment and were expected to be unable to penetrate the cell or exhibit significant cytotoxicity.
15,18,21]
GRKKRRQRRRPPQ GRKKRRQRRRPPQRQTSMTDFYHSKRRLIFS GRKKRRQRRRPPQRATSATDAYHSKRRLIFS RQTSMTDFYHSKRRLIFS RATSATDAYHSKRRLIFS
3.1. Significant cytotoxic effects can be attributed to Tat48– 60 -P10 The cytotoxic effect of each peptide at a range of 0-80 mM was investigated in cell lines derived from a DNA mismatch repair proficient colorectal cancer (SW480) and a malignant glioblastoma (U251) (Fig. 1a and b, respectively) using the MTT cell
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Fig. 2 – Cell viability comparison of the human cell lines, U251, U373, SW480 and MCF7 (as determined by MTT assay) when incubated with the cell penetrant peptide, Tat48–60-P10, and control peptides, Tat48–60, Tat48–60-P10[Ala 15,18,21], P10 and P10 [Ala 15,18,21] at a concentration of 50 mM for 24 h at 37 8C. Note the specific and relative sensitivity of U251 cells to Tat48–60-P10. Error bars indicate S.E.M. (n > 3).
viability assay [21]. At a peptide concentration of 10–20 mM, in SW480 cells no significant difference in cytotoxicity was observed between Tat48–60-P10, Tat48–60-P10[Ala 15,18,21] and P10 (Fig. 1b). However at 40 mM, Tat48–60-P10 is significantly more cytotoxic than all remaining peptides, including Tat48–60P10[Ala 15,18,21] (Mann–Whitney test, P = 0.0041) and P10 (Mann– Whitney test, P = 0.0232). At 80 mM peptide concentrations cytotoxicity that was both significant and specific to Tat48–60P10 was observed for SW480 cells. U251 cells exhibit statistically significant sensitivity to the Tat48–60-P10 peptide at a concentration of 20 mM when compared to untreated cells (Kruskal–Wallis test, P < 0.0001) whereas the control peptides (Tat48–60, Tat48–60-P10[Ala15,18,21], P10, P10[Ala15,18,21]) did not show statistically significant cytotoxicity. At 40 mM U251 cells exhibit significant sensitivity to the Tat48–60-P10 peptide (Kruskal–Wallis test, P = 0.002) but not to the control peptides. Furthermore, at a concentration of 80 mM, the Tat48–60-P10 was significantly more cytotoxic than P10, P10[Ala 15,18,21] (Dunn’s multiple comparisons test P < 0.01), but there was no longer a significant difference between Tat48–60-P10 and Tat48–60-P10[Ala 15,18,21] . Significant and specific cytotoxicity to Tat48–60-P10 was observed for SW480 cells at 80 mM peptide concentrations, whereas a trend towards non-specific cytotoxicity was observed in U251 cells (compare Fig. 1a and b). It is noteworthy that Tat-containing peptides have previously been reported to exhibit non-specific poisoning at 100 mM [33]. We then examined the effect of Tat48–60-P10 on U373 (astrocytoma) and MCF7 (breast carcinoma) cell lines, additionally incorporating an analysis of the cytotoxicity of the parent Tat48–60 peptide. Peptides were incubated at a concentration of 50 mM using the MTT cell viability assay to determine cellular sensitivity (Fig. 2). In all cell lines tested there was a significant cytotoxic effect with Tat48–60-P10 when compared to Tat48–60-P10[Ala 15,18,21], P10, P10[Ala 15,18,21] and untreated cells (Kruskal–Wallis test, P < 0.0001 with Dunn’s
multiple comparisons test, P < 0.001). Cells treated with control peptides (Tat48–60, Tat48–60-P10[Ala 15,18,21], P10, P10[Ala 15,18,21] ) showed comparable cell death to untreated cells indicating that the control peptides were not overly cytotoxic (Dunn’s multiple comparisons test, in all case P > 0.05). Significant sensitivity to Tat48–60-P10 in U251 cells when compared to SW480 (Mann–Whitney test, P < 0.0001) and MCF-7 cells (Mann–Whitney test, P < 0.0001) was notable (Fig. 2). In contrast, sensitivity in U373 cells to that of U251 cells was not significantly different (Mann–Whitney test, P = 0.1490). These data indicate that Tat48–60-P10 is specifically cytotoxic, that is, the inclusion of the PIP box sequence is necessary but insufficient without the presence of the Tat48–60 sequence for the observed cytotoxicity.
3.2. Tat48–60-P10 and Tat48–60-P10[Ala the nucleus in U251 cells
15,18, 21]
localize to
To elucidate the cell and nuclear penetrating capacity of the synthesized peptides, immunofluorescence analysis was employed using a monoclonal anti-Tat antibody and a secondary FITC conjugated antibody to observe the localization of Tat48–60-P10 and Tat48–60-P10[Ala 15,18,21] within U251 cells, which have a relatively prominent nucleus, thus facilitating the visualization of nuclear antigens (Fig. 3). Fig. 3A–C shows the weak non-specific signal visualized with antibodies in cells not incubated with the Tat containing peptides. Tat48–60 was noted to localize to the nucleus but did not show a punctate distribution (Fig. 3D–F). When cells were incubated with Tat48–60-P10[Ala 15,18,21] for 24 h, substantive fluorescence was noted within the nucleus, with localization at or near the nuclear periphery (Fig. 3G–I). In contrast, the Tat48–60-P10 peptide was observed to localize within the nucleus and exhibited a punctate distribution (Fig. 3J–L). Thus, while Tat48–60-P10[Ala 15,18,21] was incorporated into the
peptides 28 (2007) 731–740
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Fig. 3 – Localization of Tat48–60 containing peptides in U251 human glioblastoma cells. (A) Immunofluoresence analysis of U251 cells probed with mouse anti-Tat antibody (primary) and goat anti-mouse FITC (secondary) demonstrating levels of non-specific antibody staining. (B) DAPI staining of U251 cells. (C) Representation of merged image of (A) and (B) demonstrating weak non-specific signal observed with anti-Tat antibody (primary) and goat anti-mouse FITC (secondary) when the peptides had not been incubated with the cells. (D) Immunofluoresence analysis of U251 cells treated for 24 h with 50 mM of Tat48–60 at 37 8C and probed with mouse anti-Tat antibody (primary) and goat anti-mouse FITC (secondary). This demonstrates the distribution of the Tat48–60 peptide. (E) As described for (D), but demonstrating DAPI staining. (F) Merged image of (D) and (E), demonstrating that the Tat48–60 peptide localizes at the nuclear membrane and does not show a punctate distribution as observed for Tat48–60-P10. (G) Immunofluoresence analysis of U251 cells treated for 24 h with 50 mM of Tat48–60-P10[Ala 15,18,21] at 37 8C and probed with mouse anti-Tat antibody (primary) and goat anti-mouse FITC (secondary). This demonstrates the distribution of the Tat48–60-P10[Ala 15,18,21] peptide. (H) As described for (G), but demonstrating DAPI staining. (I) Merged image of (G) and (H), demonstrating the incorporation of Tat48–60-P10[Ala 15,18,21] into the cytoplasm and nucleus as indicated by the arrows. (J) Immunofluorescence analysis of U251 cells treated for 24 h with 50 mM Tat48–60-P10 at 37 8C when probed with mouse anti-Tat antibody (primary) and goat anti-mouse FITC (secondary). This demonstrates the distribution of Tat48–60-P10. (K) As described for (J), but demonstrating DAPI staining. (L) Merged image of (J) and (K), demonstrating the incorporation of Tat48–60-P10 into the cytoplasm and nucleus as indicated by the arrows. Note the increased incorporation into the nucleus of Tat48–60-P10 (J) and (L) when compared to Tat48–60 (D) and (F) Tat48–60-P10[Ala 15,18,21] (G) and (I) and the markedly different nuclear distribution. All images at T400 magnification.
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Fig. 4 – Tat48–60-P10 appreciably binds PCNA whereas Tat48–60-P10[Ala 15,18,21] does not. BSA (negative control), Tat48–60-P10[Ala 15,18,21] and Tat48–60-P10 were dotblotted onto nitrocellulose membrane, all at an equimolar concentration of 1 mM and incubated with 33 mg/mL of recombinant PCNA protein in PBS, U251 total cell extract in PBS and PBS alone for 48 h at 4 8C. PCNA binding occurred with Tat48–60-P10 but minimally with BSA or Tat48–60-P10[Ala 15,18,21]. Monoclonal anti-human PCNA (clone PC10, Sigma) primary antibody and goat anti-mouse HRP (Sigma) secondary antibody were used for immunoblotting under standard conditions. Western blot analysis of U251 cell nuclear extract and recombinant PCNA and Coomassie staining of recombinant PCNA are also shown, indicating the purity of the recombinant PCNA and confirming the presence of PCNA within U251 extracts.
nucleus, it did not show the same localization as Tat48–60-P10, which appeared to be concentrated at sites within the nucleus. Although recent evidence [4] suggests that cell fixation can lead to artificial uptake and redistribution of peptides, the differences in localization between two extremely similar peptides (Tat48–60-P10 and Tat48–60-P10[Ala 15,18,21]) and the specific cytotoxicity associated with Tat48–60-P10 suggest to us that the localization of Tat48–60-P10 is unlikely to be a fixationassociated artifact. Specific PCNA binding to Tat48–60-P10 peptide was also confirmed in-vitro in a dot-blot assay using recombinant PCNA, U251 total cell extracts (Fig. 4) and using SW480 cell nuclear extracts [36] (R.D.B. unpublished data). PCNA binding occurred with Tat48–60-P10 but minimally with BSA or Tat48–60-P10[Ala 15,18,21] indicating that Tat48–60-P10 specifically interacts with PCNA. The localization of Tat48–60P10 and PCNA within the nucleus was further investigated using immunofluorescence analysis in U251 cells (Fig. 5). In U251 cells incubated with Tat48–60-P10[Ala 15,18,21] for 24 h, while the peptide was noted in the cytoplasm and nucleus where it apparently localized at the nuclear periphery (Fig. 5B), it did not appear to colocalize with nuclear PCNA (Fig. 5C). This differential location of PCNA and Tat48–60-P10[Ala 15,18,21] can be clearly observed in merged images of Fig. 5B and C (Fig. 5D). By contrast, incubation of Tat48–60-P10 with U251 cells resulted in the nuclear localization of the peptide with a punctate distribution (Fig. 5F and J). Moreover, Tat48–60-P10 was noted to colocalize with nuclear PCNA (Fig. 5H and L), strongly supporting the contention that this peptide is capable of specifically binding PCNA in situ.
3.3.
Tat48–60-P10 is pro-apoptogenic
We hypothesized that apoptosis would be triggered as a consequence of disruption of PCNA function. We therefore performed terminal dUTP nick-end labeling (TUNEL) assays to investigate whether the cytotoxicity associated with Tat48–60P10 was due to the triggering of apoptotic pathways, and whether this was specifically associated with the addition of the PIP box sequence to the cell penetrating Tat48–60 peptide
(Fig. 6). Untreated U251 cells were TUNEL-negative (Fig. 6A–C). The TUNEL negative (TUNEL reaction mix without terminal transferase) and the TUNEL-positive (DNase 1 incubation) controls validated the assay employed (Fig. 6D–I). While after incubation with Tat48–60-P10[Ala 15,18,21], U251 cells were TUNEL-negative (Fig. 6J–L), TUNEL-positive cells were clearly noted after Tat48–60-P10 incubation (Fig. 6M–O). These findings suggest that Tat48–60-P10 specifically triggers apoptosis, rather than necrosis, as has been noted previously for the PIP box containing sequence when coupled to the anntenapedia cell penetrating peptide [22].
4.
Discussion
In this study we demonstrate that Tat48–60-P10 is cytotoxic. This toxicity is specifically related to the presence of the PIP box in the peptide construct that can bind PCNA in vitro and that can colocalize with PCNA in situ. We propose that this peptide blocks PCNA interacting with its cognate partners, triggering apoptosis. Using a range of peptide concentrations we established that Tat48–60-P10 was significantly more cytotoxic to U251 and SW480 cells than control peptides. Moreover, using a fixed peptide concentration (50 mM) we clearly demonstrate that Tat48–60-P10 is significantly cytotoxic to U373 (astrocytoma), U251 (glioblastoma), SW480 (colon adenocarcinoma), and MCF7 (breast carcinoma) cells. The use of Tat48–60-P10[Ala 15,18,21], P10, P10[Ala 15,18,21] and the Tat48–60 peptides as controls allowed us to attribute the cytotoxicity of Tat48–60-P10 to the presence of the PIP-box consensus sequence. Based on previous peptide studies, it seems reasonable to suggest that the PIP-box is interacting with PCNA. However, there are suggestions that cyclin-dependent kinase (CDK) inhibition is a key factor in preventing cell cycle progression [20,34,35]. While both Tat48–60-P10[Ala 15,18,21] and Tat48–60-P10 retain the CDK inhibitory motif of p21WAF1/CIP1 (RRLIFS) our data indicates that this is not the main factor in the differential cytotoxicity with lower concentrations used in this study, but conceivably may be a factor in the cytotoxicity
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Fig. 5 – Colocalization of Tat48–60-P10 and PCNA in U251 human glioblastoma cells. (A–D) U251 cells treated for 24 h with 50 mM Tat48–60-P10[Ala 15,18,21] at 37 8C. (A) DAPI staining; (B) localization of Tat48–60-P10[Ala 15,18,21] as indicated by anti-Tat antibody detected with FITC conjugated secondary antibody. Note the localization of Tat48–60-P10[Ala 15,18,21] at or near the nuclear periphery and in the cytoplasm. (C) Detection of PCNA (arrowed) within the nucleus using an anti-PCNA monoclonal antibody detected with rhodamine conjugated secondary antibody. (D) merged image of (B) and (C) demonstrating PCNA (indicated by the arrows) and Tat48–60-P10[Ala 15,18,21] localization. Colocalization of Tat48–60-P10[Ala 15,18,21] and PCNA was not noted. (E–L) U251 cells treated for 24 h with 50 mM of Tat48–60-P10 at 37 8C. (E) DAPI staining; (F) localization of Tat48–60-P10 as indicated by an anti-Tat antibody as in (B). Note the pattern of localization, as indicated by the arrows, of Tat48–60-P10 at specific nuclear sites. (G) Detection of PCNA within the nucleus as in (C). Arrows indicate specific areas of PCNA localization. (H) Merged image of (F) and (G) with arrows indicating the colocalization of Tat48–60-P10 with PCNA. Fig. 5(I–L) shows images at T1000 magnification. (I) DAPI staining of U251 cells; (J) localization of Tat48–60-P10 at specific sites within the nucleus (indicated with the arrows) as determined in (F). Note the different localization within the nucleus of Tat48–60-P10 when compared to Tat48–60-P10[Ala 15,18,21] compare (B) and (J). (K) PCNA detection as in (C). Arrows indicate PCNA. (L) Merged image of (J) and (K) showing that Tat48–60-P10 and PCNA colocalize within the nucleus, as indicated by the arrows. All images shown at T400 magnification unless otherwise indicated.
observed with Tat48–60-P10[Ala 15,18,21] and Tat48–60-P10 at higher concentrations (80 mM) in U251 cells. We therefore conclude that the CDK inhibitory motif alone is insufficient to cause significant cell death when the PIP-box is not conserved. Also, under the conditions described herein, at a peptide concentration of 50 mM, U251 cells appear to show increased sensitivity to Tat48–60-P10 when compared to SW480 and MCF7 but not U373 cells, suggesting differential sensitivity for cells of astrocytic lineage to Tat48–60-P10. The differing sensitivities to the Tat48–60-P10 peptide may conceivably be related to varying mechanisms of Tat uptake in different cell lines: routes of uptake of Tat containing peptides include macropinocytosis, via caveolae and endocytosis and are dependent on numerous variables [4,31]. To study the cellular penetration of the Tat-conjugated peptides immunofluorescence analysis was performed. Both
Tat48–60-P10 and Tat48–60-P10[Ala 15,18,21] were incorporated into the nucleus within 24 h. Furthermore, the possession of the PIP-box sequence, and the noted punctate distribution of Tat48–60-P10 within the nucleus, in comparison to Tat48–60P10[Ala 15,18,21], led us to propose that Tat48–60-P10 was associating with PCNA at replication foci. Prior to the commencement of DNA replication PCNA localizes to prereplicative sites where recruitment of replication factors is thought to occur [17,25,35]. Many proteins containing the PIPbox associate with PCNA in replication foci during S-phase of the cell cycle [34], so it is perhaps not surprising that Tat48–60P10, containing a PIP box, would also associate at replication foci. While recent findings have suggested that Tat48–60 can be rapidly degraded [30], it is formally possible that Tat48–60-P10 is in a stable complex with PCNA and unavailable for degradation whereas Tat48–60-P10[Ala 15,18,21] is more readily subjected
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Fig. 6 – Tat48–60-P10 incubation in U251 human glioblastoma cells induces apoptosis. (A) TUNEL analysis of untreated U251 cells visualized with Texas red indicating that apoptosis has not been triggered; (B) as (A) but demonstrating DAPI staining; (C) representation of merged image of (A) and (B) demonstrating a TUNEL negative result with untreated U251 cells. (D) Texas red visualization of a TUNEL negative control where the TUNEL reaction mix does not contain terminal transferase; (E) DAPI stain for TUNEL negative control; (F) representation of merged image of (D) and (E) demonstrating a TUNEL negative result for the negative control. (G) Texas red visualization of a TUNEL positive control (employing DNase I) which catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone allowing nick-end labeling to occur; (H) DAPI stain for TUNEL positive control; (I) merged image of (G) and (H) demonstrating a TUNEL positive result for the positive control. (J)
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to proteolytic cleavage. It is worth stating that Tat48–60 uptake is a one-way process leading to the trapping of the fragment within the cell as Tat48–60 induces minimal membrane leakage [13,38]. While most Tat48–60-P10 was seen to penetrate into the nucleus, cytoplasmic fluorescence was also evident. As PCNA can be localized within several sub-cellular compartments [23], this extra-nuclear fluorescence may represent Tat48–60P10 binding cytoplasmic PCNA. Although fixation of cells can lead to artificial incorporation of cationic peptides such as Tat48–60 [29], recent optical and biophysical analysis of live fibroblast uptake of the Tat cell penetrating peptide sequence has substantively confirmed the nuclear penetrating properties of the peptide [37]. We, therefore, conclude that the observed focal localization of Tat48–60-P10 is unlikely to be a fixation-associated artifact. Colocalization studies consistently demonstrated that Tat48–60-P10 and PCNA could be found at similar positions within the nucleus. This was in contrast to Tat48–60-P10[Ala 15,18,21] which, although noted to enter the nucleus, failed to colocalize with PCNA. Support for Tat48–60-P10-PCNA binding to PCNA also comes from the dotblot analysis undertaken: Tat48–60-P10 was specifically able to bind PCNA, while Tat48–60-P10[Ala 15,18,21] and Tat48–60 alone was unable to bind PCNA. The biological activity of Tat48–60-P10 was characterized using a TUNEL assay to investigate the mechanism of cell death triggered after incubation with Tat48–60-P10. Surprisingly, in contrast to previous findings of induction of necrosis (assessed by electron microscopy) in a human lymphoma cell line treated with a p21WAF1/CIP1 derived peptide coupled to the anntenapedia peptide sequence (W10-AP) [22] we noted that Tat48–60-P10 triggered apoptosis in U251 cells whereas the control peptide Tat48–60-P10[Ala 15,18,21] – at the same concentration – did not. The ability of Tat48–60-P10 to trigger apoptosis, in comparison to the reported necrosis with the W10-AP peptide [22], may be due to the orientation of the cargo. The p21 PIP box, unlike W10-AP, was coupled by the C-terminal to the N-terminal of Tat which may have decreased the cellular uptake of the Tat48–60-P10 peptide [26] but potentially allowed the p21 PIP box the freedom to bind specifically to PCNA (and possibly CDK-cyclin complexes) more effectively, thereby triggering apoptosis rather than necrosis. The W10-AP peptide was considerably larger than Tat48–60-P10 and still retained the bipartite nuclear localisation sequence of p21, which was negated by Tat48–60 in the Tat48–60-P10 peptide [22]. The cytotoxicity of p21 derived peptides conjugated to anntenapedia has previously been established in two human ovarian cell lines [2], however, apoptosis was not reported after exposure to these particular peptides and the inhibition of growth appeared related to disruption of CDK2 and cdc2 function [2]. Another p21 based peptide, known as Tat-p21PBD, designed to associate with CDKs, has been shown to accumulate in the nucleoli of cells and it has been theorized
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that the high intracellular mobility of Tat containing peptides make them useful for creating biological effects [32]. After treatment with Tat-p21-PBD, only 40% of cells entered S-phase perhaps suggesting that Tat48–60-P10 may inhibit S phase entry [32], nevertheless, the punctuate distribution of Tat48–60-P10 suggests an association with PCNA at replication foci during Sphase. A cytotoxic Tat-p21 fusion protein containing the PIP box region [7] was theorized to inhibit PCNA function, although this was not proven. Herein, it has been proven that Tat48–60-P10 does indeed colocalize with PCNA and binding studies clarify that the peptide binds to PCNA specifically whereas controls do not. During cell viability experiments membrane blebbing was seen to occur with all cell lines treated with Tat48–60-P10 but not with Tat48–60-P10[Ala 15,18,21] , P10 and P10[Ala 15,18,21] (R.D.B., unpublished observations). These findings suggest that interference of PCNA mediated by the PIP-box containing peptide can trigger apoptotic as well as necrotic pathways and firmly suggests that the cytotoxicity observed with Tat48–60-P10 is due to the disruption of PCNA function.
Acknowledgements We wish to thank Keith Holding, Harpreet Dibra and Sarah Jones for technical assistance. We are extremely grateful to C. Ludwig and M. Walkinshaw, University of Edinburgh for the generous gift of recombinant PCNA. This study was supported by intramural funding from the University of Wolverhampton.
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TUNEL analysis of U251 cells treated for 24 h with 50 mM Tat48–60-P10[Ala 15,18,21] at 37 8C visualized with Texas red; (K) as described for (H), DAPI staining; (L) the merged image of (J) and (K), demonstrating a TUNEL negative result for cells treated with Tat48–60-P10[Ala 15,18,21], indicating that the peptide lacking the complete PIP-box does not trigger apoptosis. (M) TUNEL analysis of U251 cells treated for 24 h with 50 mM Tat48–60-P10 at 37 8C visualized with Texas red, demonstrating that Tat48–60-P10 triggers apoptosis; (N) as described for (K), DAPI staining; (O) merged image of (M) and (N), indicative of a TUNEL positive result for Tat48–60-P10. Thus, Tat48–60-P10 (M–O) triggers apoptosis in U251 cells whereas Tat48–60-P10[Ala 15,18,21] (J–L) does not, indicating that the p21 PIP box sequence in combination with Tat48–60 is essential for triggering programmed cell death.
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