Inhibition of Cell Proliferation by the PCNA-Binding Region of p21 Expressed as a GFP Miniprotein

doi:10.1006/excr.2001.5160

Experimental Cell Research

Volume 265, Issue 2, 1 May 2001, Pages 234-241

https://doi.org/10.1006/excr.2001.5160 Get rights and content

Abstract

p21 (WAF1/Cip1) is the only member of the CIP/KIP family which has a well-characterized PCNA-binding domain. p21 is known to have an important function in the coordination of the cellular pathways which are activated in response to DNA damage, though the significance of the p21–PCNA interaction is not completely clear. We have analyzed the effects of expressing a miniprotein containing the PCNA-binding domain of p21 upon the cell cycle and upon the proliferation of various cell types. We have compared this with the effect of expressing a mutant form which is defective in PCNA-binding, but which retains the secondary cyclin–CDK-inhibitory site. No PCNA-dependent effects were seen in the short term upon cell cycle distribution. However, clonogenic assays show that the GFP-peptide miniprotein can significantly suppress proliferation in a PCNA-dependent manner. In some cell types, however, the suppression of proliferation was not PCNA-dependent, suggesting that cellular environment is a contributory factor to the effect of this miniprotein. The capacity of this peptide sequence to suppress cell proliferation in vivo is of interest as the basis for the design of potential antiproliferative therapeutic agents.

References (24)

E. Warbrick et al.
A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen
Curr. Biol.
(1995)
J.M. Gulbis et al.
Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA
Cell
(1996)
M.K.K. Shivji et al.
CIP1 inhibits DNA replication but not PCNA dependent nucleotide excision repair
Curr. Biol.
(1994)
R. Li et al.
Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage
Curr. Biol.
(1996)
M.T. Scott et al.
Reversible phosphorylation at the C-terminal regulatory domain of p21(WAF1/CIP1) modulates proliferating cell nuclear antigen binding
J. Biol. Chem.
(2000)
K.L. Ball et al.
Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21(WAF1)
Curr. Biol.
(1997)
S. Waga et al.
The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA
Nature
(1994)
R. Li et al.
Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair
Nature
(1994)
H. Flores-Rozas et al.
Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme
Proc. Natl. Acad. Sci. USA
(1994)
S. Waga et al.
Cyclin-dependent kinase inhibitor p21 modulates the DNA primer–template recognition complex
Mol. Cell. Biol.
(1998)

M.K. Shivji et al.

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

Oncogene

(1998)

Cited by (31)

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions
2017, DNA Repair
Citation Excerpt :
In PCNAS228I cells the reduced affinity for other PIP-box proteins would mean that p21 is even more likely to gain control of the PCNA binding interface. Ectopic expression of high affinity, non-degradable PIP-box-containing fusion proteins or peptides perturbs the equilibrium of PCNA interactions and has profound effects on cell proliferation [84,85]. Of course, p21 contains a PIP degron and normally the PCNA associated p21 is efficiently degraded, clearing the way for subsequent association of the next client protein.
Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA^S228I mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3^p66 and PolD4^p12. In contrast p21 largely retains the ability to bind PCNA^S228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA^S228I binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA^S228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.
Anti-tumor efficacy of a therapeutic peptide based on thermo-responsive elastin-like polypeptide in combination with gemcitabine
2014, Cancer Letters
Citation Excerpt :
p21Waf1/Cip1, a member of the CIP/KIP family of cyclin dependent kinase, has a PCNA-binding domain at the C-terminus and a cyclin dependent kinase (CDK)-inhibitory motif at the N-terminus [20]. As p21 mimics the C-terminus of p21, it controls CDK activity and inhibits PCNA functions, thereby leading to arrest at the G1 and G2 or S phase of the cell cycle [20,21]. Well-expressed p21-ELP1-Bac and its scrambled control (scr p21-ELP1-Bac) were examined to determine their transition temperatures.
This work describes the effects of elastin-like polypeptide (ELP) with the p21^Waf1/Cip1-derived cell cycle inhibitory peptide (p21) on pancreatic tumor cells with gemcitabine. The thermo-responsive property of ELP permits use of a mild, local hyperthermia to target tumors for the transport of chemotherapeutics. In this study, a p21-ELP construct with Bac cell penetrating peptide was designed, and its anticancer activities in pancreatic cancer cell lines was examined. In combination with gemcitabine, the peptide demonstrated enhanced in vitro cytotoxicity as well as tumor growth inhibition in an animal model. Our data suggest that this ELP construct, with gemcitabine, may improve pancreatic cancer therapy.
Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay
2012, Analytical Biochemistry
Citation Excerpt :
Several studies used the p21 peptide as an anticancer agent and showed that p21 peptides fused to cell-penetrating peptide tags displayed significant antiproliferative activity against human keratinocyte-derived HaCaT cells, DLD1 colon cancer cells, and CA46 lymphoma cells [38–41]. Similarly, a GFP-fused p21 peptide inhibited cell proliferation of H1299 non-small-cell lung carcinoma cells, U2OS osteosarcoma cells, and Saos2 osteocarcinoma cells [42]. Despite efforts to improve the potency of the p21-based PCNA antagonist, few peptides have shown better affinity to PCNA compared with the native p21 peptide [21].
Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed, the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work, we report a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. To improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we show that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared with the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.
Cell penetrating elastin-like polypeptides for therapeutic peptide delivery
2010, Advanced Drug Delivery Reviews
Citation Excerpt :
Therefore, the use of the full length 141–160 peptide as an inhibitor may lead to two separate mechanisms of action. These peptides, when fused to the penetratin CPP, have shown antiproliferative activity against human keratinocyte-derived HaCaT cells [75], DLD1 colon cancer cells [76], and CA46 lymphoma cells [77]; and a GFP-fused p21 peptide inhibited proliferation of H1299 non-small cell lung carcinoma cells (p53 deletion), U2OS osteosarcoma cells (p53 wild type), and Saos2 osteocarcinoma cells (p53 deletion) [78]. In order to facilitate targeted delivery, we fused the p21 139–164 peptide to the C-terminus of ELP.
Current treatment of solid tumors is limited by side effects that result from the non-specific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer.
Multiple roles of the cell cycle inhibitor p21<sup>CDKN1A</sup> in the DNA damage response
2010, Mutation Research - Reviews in Mutation Research
Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21^CDKN1A plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation.
In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response.
Ultraviolet B and A irradiation induces fibromodulin expression in human fibroblasts in vitro
2009, Biochimie
Citation Excerpt :
To identify cells in replicative senescence, we determined the levels of p21waf1 mRNA and the number of SA-βgal (senescence associated-β-galactosidase activity)-positive cells. p21waf1 plays an important role in the regulation of cellular senescence because this cyclin-dependent kinase inhibitor (CDKI) binds to “cyclin-CDK” complexes and causes cell cycle arrest in the G1/S phase [19]. As shown in Fig. 3A,C, UVB-irradiation at doses between 10 and 60 mJ/cm2 provoked a gradual increase of p21waf1 mRNA and a concomitant increase of SA β-gal-positive cells (about 50% at 60 mJ/cm2 UVB irradiation).
Ultraviolet (UV) radiation affects the extracellular matrix (ECM) of the human skin. The small leucine-rich repeat protein fibromodulin interacts with type I and II collagen fibrils, thereby affecting ECM assembly. The aim of this study was to evaluate whether short wave UV (UVB) or long wave UV (UVA) irradiation influences fibromodulin expression. Exponentially growing human fibroblasts (IMR-90 cells) were exposed to increasing doses of UVB (2.5–60 mJ/cm²) or UVA (0.5–10 J/cm²). After UV irradiation fibromodulin, p21 and GADD45 levels were evaluated as well as cell viability, reactive oxygen species formation (ROS) and DNA damage. We found that fibromodulin expression: (i) increased after UVB and UVA irradiation; (ii) was 10-fold higher after UVA (10 J/cm²) versus 5-fold with UVB (10 mJ/cm²); (iii) correlated with reactive oxygen species formation, particularly after UVA; and (iv) was linked to the DNA damage binding protein (DDB1) translocation in the nucleus, particularly after UVB. These results further suggest that the UV-induced fibromodulin increase could counteract the UV-induced connective tissue damage, promoting the assembly of new collagen fibrils.

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¹: To whom correspondence and reprint requests should be addressed at the Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. Fax: (01382) 496 363. E-mail: [email protected].

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Regular ArticleInhibition of Cell Proliferation by the PCNA-Binding Region of p21 Expressed as a GFP Miniprotein

Abstract

Curr. Biol.

Cell

Curr. Biol.

Curr. Biol.

J. Biol. Chem.

Curr. Biol.

The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA

Nature

Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair

Nature

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme

Proc. Natl. Acad. Sci. USA

Cyclin-dependent kinase inhibitor p21 modulates the DNA primer–template recognition complex

Mol. Cell. Biol.

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

Oncogene

Regular Article
Inhibition of Cell Proliferation by the PCNA-Binding Region of p21 Expressed as a GFP Miniprotein