Kit Contents:

Component	Specification	Concentration (total volume)
p53 Reporter	A mixture of inducible p53-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

General performance

Average maximum response rate = 117
Average Z' factor at maximum response rate = 0.73
Average coefficient of variation (CV%) = 9.2%

Excellent signal to noise ratio

Figure 1: Cignal p53 reporter assay showed upregulation of p53 signaling activity: 293-H cells were transfected with p53 reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated separately with 100µM Nutlin (a small chemical and well-known MDM-2 antagonist) or DMSO alone. Dual Luciferase assay was performed 18 hours after treatment, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal p53 reporter assay measured 260 fold increase in p53 transcription activity by 100 µM nutlin.

Excellent specificity

Figure 2: Cignal p53 reporter showed downregulation of p53 transcription activity: HCT 116 cells were transfected with p53 reporter, negative control and positive control along with p53 siRNA or negative control siRNA (for transfection protocol refer our user manual). After 24 hours of transfection, medium was changed to complete medium. Dual Luciferase assay was performed 72 hours after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal p53 reporter measured 75% abolishment of p53 transcription activity by p53 siRNA..

p53 is a tumour suppressor protein that regulates the expression of a wide variety of genes involved in Apoptosis, Growth arrest, Inhibition of cell cycle progression, Differentiation and accelerated DNA repair or Senescence in response to Genotoxic or Cellular Stress. As a transcription factor, p53 is composed of an N-terminal Activation Domain, a central specific DNA Binding Domain, and a C-terminal Tetramerization Domain, followed by a Regulatory Domain rich in basic Amino acids. Having a short half-life, p53 is normally maintained at low levels in unstressed mammalian cells by continuous ubiquitylation and subsequent degradation by the 26S Proteasome. Nonphosphorylated p53 is ubiquitylated by the MDM2 (Mouse Double Minute-2) ubiquitin ligase. MDM2 binding inactivates p53 by two mechanisms. First, MDM2 binds to the transactivation domain of p53, precluding interaction with the transcriptional machinery. Second, this binding mediates the covalent attachment of ubiquitin to p53. Ubiquitylated p53 is then degraded by the Proteasome. Thus MDM2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. When the cell is confronted with stress like DNA damage, Hypoxia, Cytokines, Metabolic changes, Viral infection, or Oncogenes, however, p53 ubiquitylation is suppressed and p53 accumulates in the nucleus, where it is activated and stabilized by undergoing multiple covalent modifications including Phosphorylation and Acetylation (Ref.1 & 2).

Phosphorylation of p53 mostly occurs in the N-terminal activation domain at the Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, and Thr81 residues, with some phosphorylation occurring in the C-terminal linker and basic regions at Ser315, Ser371, Ser376, Ser378, and Ser392. Phosphorylation on most of these sites is induced by DNA damage, with some, such as Thr55 and Ser376, being repressed upon genotoxic stress. p53 phosphorylation is mediated by several cellular kinases including Chks (Checkpoint Kinases), CSNK1-Delta (Casein Kinase-1-Delta), CSNK2 (Casein Kinase-2), PKA (Protein Kinase A), CDK7 (Cyclin-Dependent Kinase-7), DNA-PK (DNA-Activated- Protein Kinase), HIPK2 (Homeodomain-Interacting Protein Kinase-2), CAK (CDK-Activating Kinase), p38 and JNK (Jun NH2-terminal kinase). Notably, phosphorylation at Ser15 by ATM (Ataxia Telangiectasia Mutated Gene)/ATR (Ataxia-Telangiectasia and Rad3 Related), either directly or through Chk1 (Cell Cycle Checkpoint Kinase-1)/Chk2 (Cell Cycle Checkpoint Kinase-2), or at Ser20 by Chk1/Chk2 has been shown to alleviate the inhibition or degradation of p53, leading to p53 stabilization and activation. The phosphorylation-induced p53 stabilization and activation are mediated through multiple mechanisms and may vary according to the cellular context or microenvironment. HIF-1Alpha (Hypoxia-Inducible Factor-1-Alpha) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1Alpha regulates p53-mediated function remains unknown. Recently, the interaction between p53 and HIF-1Alpha was reported to evoke HIF-1Alpha degradation. Members of the PIAS (Protein Inhibitor of Activated STAT) protein family have also been found to interact with p53. PIAS1 and PIAS-Gamma function as SUMO (Small Ubiquitin Related Modifier-1) ligases for p53. Moreover, the RING finger domain of PIAS1 binds to the C-terminus of the tumor suppressor p53 and catalyzes its sumoylation, a modification which represses p53 activity on a reporter plasmid containing consensus p53 DNA binding sites. PML (Promyelocytic Leukemia) also activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. PML is a tumor suppressor protein and the major component of multiprotein nuclear complexes that have been variably termed Kremer bodies, ND10, PODs (for PML Oncogenic Domains), and PML-NBs (PML-Nuclear Bodies). PML binds directly with p53 and recruits it to PML-NBs. Recruitment to PML-NBs activate p53 by bringing it in close proximity with CBP (CREB-Binding Protein) /p300. BRCA1 (Breast Cancer-1 Gene) and p53 can also physically associate, both in vitro and in vivo and function in a common pathway of tumor suppression. The ability of BRCA1 to biochemically modulate p53 function suggests that this may be a fundamental role of BRCA1 in tumor suppression (Ref.3, 4 & 5).

Another important modification of p53 is acetylation. p53 is specifically acetylated at Lys370, Lys372, Lys373, Lys381, and Lys382 by p300/CBP and at Lys320 by PCAF (p300/CBP-associated factor). Acetylation has been shown to augment p53 DNA binding, and to stimulate p53-mediated transactivation of its downstream target genes through the recruitment of coactivators. Acetylation may also regulate the stability of p53 by inhibiting its ubiquitination by MDM2. In vivo, acetylation at Lys320, Lys373, and Lys382 is induced by many genotoxic agents, including UV-radiation, IR (Ionizing Radiation), hypoxia, oxidative stress, and even depletion of ribonucleotide pools. p53 can also be deacetylated by HDAC1 (Histone Deacetylase-1) and SIRT1. Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. p53 deacetylation has been suggested to down-regulate the activation of genes such as Bax and p21WAF1. Phosphorylation and acetylation are interdependent. Indeed, phosphorylation at the p53 N-terminus has been shown to enhance its interaction with acetylase p300/CBP and to potentiate p53 acetylation. Activated p53 functions effectively as a transcription factor and induces transcription of several genes. The DNA targets of p53 are consensus sequences consisting of a 10-base pair repeat of 5'-PuPuPu-C(A/T)(T/A)GPyPyPy-3' (where Pu is a purine and Py is a pyrimidine). It also can bind to a palindromic site having a four or five-base pair inverted repeat of a similar sequence. Complete p53 is inactive for specific DNA binding unless activated by covalent and noncovalent modifications of the basic C-terminal domain. After p53 is activated it can be involved in cell-cycle inhibition, apoptosis, genetic repair, and inhibition of blood-vessel formation (Ref.5, 6 & 7).

Cell cycle inhibition takes place when there is a block in cell-cycle division. p53 does this by stimulating the expression of p21 WAF1/CIP1 (Cyclin Dependent Kinase Inhibitor-p21). This protein is an inhibitor of CDKs (Cyclin-Dependent Kinases) that regulate the cell cycle via perturbation of their partner cyclin. Cyclins are involved to ensure successful transitions from S phase to G1. Since p21 WAF1/CIP1 inhibits CDKs it results in inhibition of both G1-to-S and G2-to-mitosis transitions by causing hypophosphorylation of Rb (Retinoblastoma) and preventing the release of E2F. Additionally p53 can stimulate 14-3-3, a protein that sequesters Cyclin B1-CDK1 complexes out of the nucleus. This results in a G2 block. Activated p53 may also initiate apoptosis and stop cell proliferation. p53 stimulates a wide network of signals that act through two major apoptotic pathways: Extrinsic Pathways and Intrinsic Pathways. The extrinsic pathway involves engagement of particular `death' receptors that belong to the TNFR (Tumor Necrosis Factor Receptor) family and, through the formation of the DISC (Death-Inducing-Signaling-Complex), leads to a cascade of activation of Caspases, including Caspase8 and Caspase3, which in turn induce apoptosis. Most common death receptors involved in extrinsic apoptosis Fas, DR5 (Death Receptor-5) and PERP. The intrinsic apoptotic pathway is dominated by the Bcl2 (B-Cell CLL/Lymphoma-2) family of proteins, which governs the release of CytoC (Cytochrome-C) from the mitochondria. The Bcl2 family comprises anti-apoptotic (pro-survival) and pro-apoptotic members. The Bcl2 family is divisible into three classes: pro-survival proteins, whose members are most structurally similar to Bcl2, such as BclXL; pro-apoptotic proteins, BAX (Bcl2 Associated-X Protein) and BAK (Bcl2 Antagonist Killer-1), which are structurally similar to Bcl2 and BclXL and antagonize their pro-survival functions; and the pro-apoptotic `BH3-only' proteins. Intriguingly, a key subset of the Bcl2 family genes are p53 targets, including BAX, Noxa, PUMA (p53-Upregulated Modulator of Apoptosis) and the most recently identified, BID (BH3 Interacting Domain Death Agonist). p53 may also inhibit Bcl2 that is an inhibitor of apoptosis. p53 may also have a role in maintaining genetic stability by 'nucleotide-excision' repair of DNA, chromosomal recombination and chromosome segmentation. GADD45 (Growth Arrest- and DNA Damage-Inducible Gene-45) is a multifunctional protein that is regulated by p53 and that may play a role in DNA repair and cell cycle checkpoints. p53 can play a role in the inhibition of blood-vessel formation. In order for tumours to reach a large size, they must initiate the growth of nutrient-bringing blood vessels in their vicinity, the process of angiogenesis. p53 stimulates the production of genes that prevent this process from happening. p53 activates the expression of the Tsp1 (Thrombospondin-1), an anti-angiogenic factor, along with other angiogenesis inhibitor BAI1 (Brain-specific Angiogenesis Inhibitor-1) (Ref. 8, 9 & 10).

In addition, p53 regulates MDM2 function in a negative feedback loop, because the MDM2 gene is a target for p53. Therefore, activation of p53 eventually leads to its own inactivation by switching on a pathway that leads to its destruction. MDM2 is subject to further regulation by direct binding of the ARF (Active Response Factor) protein, which prevents MDM2-mediated p53 proteolysis. PTEN (Phosphatase and Tensin Homolog), on the other hand inhibits MDM2-mediated p53 degradation. p53 can transcriptionally activate PTEN, which may further inhibit Akt activity. Therefore, inhibition of Akt by the inhibitors may trigger a positive feedback with perhaps additional anti-tumor effects. The c-Fos proto-oncogene is also a target for transactivation by the p53 tumor suppressor. Mutations in p53 are associated with genomic instability and increased susceptibility to cancer. It is the most frequently mutated protein in all cancer with an estimated 60% of all cancers having mutated forms that affect its growth suppressing activities. However some common tumours have a higher incidence such that 90% of cervical and 70% of colorectal are found to have p53 mutations. The p53 protein can be inactivated in several ways, including inherited mutations that result in a higher incidence of certain familial cancers such as Li-Fraumeni syndrome. Certain DNA tumour viruses, such as the human adenovirus and the papilomavirus, bind to and inactivate the protein. Functional p53 is thought to provide a protective effect against tumorigenesis (Ref.2, 11 & 12).

References:

1. Hanazono K, Natsugoe S, Stein HJ, Aikou T, Hoefler H, Siewert JR.
Distribution of p53 mutations in esophageal and gastric carcinomas and the relationship with p53 expression.
Oncol Rep. 2006 Apr;15(4):821-4.

2. Francoz S, Froment P, Bogaerts S, De Clercq S, Maetens M, Doumont G, Bellefroid E, Marine JC.
Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating and quiescent cells in vivo.
Proc Natl Acad Sci U S A. 2006 Feb 21;
PubMed ID: 16492744

3. Ho CC, Siu WY, Lau A, Chan WM, Arooz T, Poon RY.
Stalled replication induces p53 accumulation through distinct mechanisms from DNA damage checkpoint pathways.
Cancer Res. 2006 Feb 15;66(4):2233-41.
PubMed ID: 16489026

4. Bianchi G, Di Giulio C, Rapino C, Rapino M, Antonucci A, Cataldi A.
p53 and p66 Proteins Compete for Hypoxia-Inducible Factor 1 Alpha Stabilization in Young and Old Rat Hearts Exposed to Intermittent Hypoxia.
Gerontology. 2006;52(1):17-23.
PubMed ID: 16439820

5. Navaraj A, Mori T, El-Deiry WS.
Cooperation Between BRCA1 and p53 in Repair of Cyclobutane Pyrimidine Dimers.
Cancer Biol Ther. 2005 Dec;4(12):1409-14.
PubMed ID: 16357511

6. Ceskova P, Chichger H, Wallace M, Vojtesek B, Hupp TR.
On the Mechanism of Sequence-specific DNA-dependent Acetylation of p53: The Acetylation Motif is Exposed upon DNA Binding.
J Mol Biol. 2006 Mar 24;357(2):442-56.
PubMed ID: 16438982

7. Kim IA, Shin JH, Kim IH, Kim JH, Kim JS, Wu HG, Chie EK, Ha SW, Park CI, Kao GD.
Histone deacetylase inhibitor-mediated radiosensitization of human cancer cells: class differences and the potential influence of p53.
Clin Cancer Res. 2006 Feb 1;12(3 Pt 1):940-9.
PubMed ID: 16467109

8. Zheng QH, Ma LW, Zhu WG, Zhang ZY, Tong TJ.
p21(Waf1/Cip1) plays a critical role in modulating senescence through changes of DNA methylation.
J Cell Biochem. 2006 Mar 2; [Epub ahead of print]
PubMed ID: 16514663

9. Akhtar RS, Geng Y, Klocke BJ, Roth KA.
Neural precursor cells possess multiple p53-dependent apoptotic pathways.
Cell Death Differ. 2006 Mar 3;
PubMed ID: 16514420

10. Rak J, Milsom C, May L, Klement P, Yu J.
Tissue Factor in Cancer and Angiogenesis: The Molecular Link between Genetic Tumor Progression, Tumor Neovascularization, and Cancer Coagulopathy.
Semin Thromb Hemost. 2006 Feb;32(1):54-70.
PubMed ID: 16479463

11. Gomes CP, Andrade LA.
PTEN and p53 expression in primary ovarian carcinomas: immunohistochemical study and discussion of pathogenetic mechanisms.
Int J Gynecol Cancer. 2006 Jan-Feb;16 Suppl 1:254-8.
PubMed ID: 16515600

12. Jiang PH, Motoo Y, Garcia S, Iovanna JL, Pebusque MJ, Sawabu N.
Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer.
World J Gastroenterol. 2006 Feb 7;12(5):691-6.
PubMed ID: 16521180



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