An immunochemical analysis of the human nuclear phosphoprotein p53: New monoclonal antibodies and epitope mapping using recombinant p53

doi:10.1016/0022-1759(92)90122-A

Journal of Immunological Methods

Volume 151, Issues 1–2, 6 July 1992, Pages 237-244

https://doi.org/10.1016/0022-1759(92)90122-A Get rights and content

Abstract

Somatic mutation of the p53 gene is a very frequent event in the development of human neoplasia, and germ line mutations in p53 are responsible for an inherited cancer susceptibility syndrome. Many of the mutations in p53 found in human tumours are point mutations that result in the substitution of a single amino acid in the protein. These point mutant proteins are much more stable than the normal protein and the mutant product accumulates to a high level which permits important information about p53 expression to be obtained by immunochemical analysis. Using bacterial expression systems to produce fragments of human p53 we have isolated and characterised new monoclonal antibodies to p53. These antibodies are suitable for the measurement of p53 in ELISA, immunoblotting and immunoprecipitation analyses. They are especially useful in immunohistochemistry as they are able to react strongly with p53 in conventionally fixed and processed histological sections.

References (24)

C.A. Finlay et al.
The p53 protooncogene can act as a suppressor of transformation
Cell
(1989)
P.A. Hall et al.
p53 immunostaining is a marker of malignancy in diagnostic cytopathology
Lancet
(1991)
R. Iggo et al.
Increased expression of mutant forms of p53 oncogene in primary lung cancer
Lancet
(1990)
R. Thomas et al.
Characterization of human p53 antigens employing primate specific monoclonal antibodies
Virology
(1983)
S.J. Baker et al.
Suppression of human colorectal carcinoma cell growth by wild-type p53
Science
(1990)
L. Banks et al.
Isolation of human p53 specific monoclonal antibodies and their use in the studies of human p53 expression
Eur. J. Biochem.
(1986)
J. Bartek et al.
Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines
Oncogene
(1990)
J. Bartek et al.
Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies
Oncogene
(1991)
W.P. Bennett et al.
Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer
Oncogene
(1991)
G. Cattoretti et al.
p53 expression in breast cancer
Int. J. Cancer
(1988)

P.-L. Chen et al.

Genetic mechanism of tumor suppression by the human p53 gene

Science

(1990)

J.V. Gannon et al.

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form

EMBO J.

(1990)

Cited by (583)

p53 Expression in Luminal Breast Cancer Correlates With TP53 Mutation and Primary Endocrine Resistance
2023, Modern Pathology
TP53 mutation is associated with primary endocrine resistance in luminal breast cancer (BC). Nuclear accumulation of p53, as determined by immunohistochemistry (IHC), is a surrogate marker for TP53 mutation. The immunohistochemical p53 index that defines a p53-positive status is not well established. This study determined the optimal p53 index cutoff to identify luminal BCs harboring TP53 mutations. In total, 364 luminal BCs from the West German Study Group ADAPT trial (NCT01779206) were analyzed for TP53 mutations by next-generation sequencing and for p53 expression by IHC (DO-7 antibody). P53 indices were determined by automated image analysis. All tumors were from patients treated with short-term preoperative endocrine therapy (pET; tamoxifen or aromatase inhibitor) before tumor resection. IHC evaluation included needle biopsies before therapy (baseline) and resections specimens after therapy (post-pET). Optimal p53 index cutoffs were defined with Youden statistics. TP53 mutations were detected in 16.3% of BC cases. The median p53 indices were significantly higher in TP53-mutated BCs compared to BCs harboring wild-type TP53 (baseline: 47.0% vs 6.4%, P < .001; post-pET: 50.1% vs 1.1%, P < .001). Short-term pET decreased p53 indices in BCs harboring wild-type TP53 (P < .001) but not in TP53-mutated BCs (P = .102). For baseline biopsies, the optimal p53 index cutoff was ≥34.6% (specificity 0.92, sensitivity 0.63, Youden index 0.54, accuracy: 0.87). For post-pET specimens, the optimal cutoff was ≥25.3% (specificity 0.95, sensitivity 0.65, Youden index 0.60, accuracy: 0.90). Using these cutoffs to define the p53 status, p53-positive BCs were >2-fold more common in pET nonresponders compared to pET responders (baseline: 37/162, 22.8% vs 18/162, 11.1%, P = .007; post-pET: 36/179, 20.1% vs 16/179, 8.9%, P = .004). In summary, IHC for p53 identifies TP53-mutated luminal BCs with high specificity and accuracy. Optimal cutoffs are ≥35% and ≥25% for treatment-naïve and endocrine-pretreated patients, respectively.
A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
2022, Structure
Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT^∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT^∗ domain. We conclude that interactions with NT^∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT^∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
Dual redox labeling of DNA as a tool for electrochemical detection of p53 protein-DNA interactions
2019, Analytica Chimica Acta
Citation Excerpt :
This type of binding is performed by a basic segment of the protein C-terminus (C-terminal DNA binding site, CTDBS) [32]. Use of monoclonal antibodies whose epitopes coincide with the CTDBS (such as Bp53–10.1) enables to modulate the mode of p53 DNA binding in the sense of blocking the CTDBS, which disables the structure-selective DNA binding of p53 but enhances its ability to bind DNA in a sequence-specific manner [33,37]. Thus, apart from the p53 importance in organisms’ defense against cancer development, its ability to bind DNA in various modes, ranging from non-specific interactions to sequence-specific DNA binding, makes it a convenient model protein for development of a novel approach for dual detection of protein-DNA interactions based on redox-labeled DNA probes.
We present a novel dual redox labeling approach enabling a facile relative evaluation of protein-DNA interactions based on immunoprecipitation at magnetic beads (MBIP) with subsequent electrochemical detection. DNA probes labeled with two different electroactive markers, benzofurazane and nitrobenzene, which yield reduction peaks at distinct potentials, were synthesized using primer extension (PEX) reaction. We show that using the labeled DNA probes, specific and non-specific binding of the p53 protein can be distinguished in a simple competition binding experiment, as a strong preference of the p53 protein was observed towards DNA probes bearing a specific p53 binding site (p53CON), which is in agreement with known binding properties of the p53 protein. The p53 binding to the individual DNA probes can be modulated by specific monoclonal antibodies used for the immunoprecipitation. This approach can potentially be applied, after selection of appropriate DNA probes and monoclonal antibodies, for investigations of DNA-binding properties of other proteins and thus represents a versatile tool for studies of any DNA-binding proteins.
Expression of cell cycle proteins according to HPV status in oral squamous cell carcinoma affecting young patients: a pilot study
2018, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Citation Excerpt :
Tumor suppressor proteins negatively modulate neoplastic transformation, inhibiting progression through cell cycle checkpoints, where they exhibit changes in DNA and others promote apoptosis.32 The tumor suppressor protein p53 has the regulatory function of avoiding duplication of damage to DNA that regulates the cell cycle, DNA repair, and apoptosis, as well as inhibiting tumor formation.38 Protein p16 is encoded by the gene CDKN2 A CDK, which acts by binding to CDK4/CDK6, preventing their interaction with cyclin D.
Tobacco and alcohol consumption are considered the main risk factors for oral squamous cell carcinoma (OSCC); however, the role of these factors in patients younger than 40 years is controversial, so it has been suggested that genomic instability and high-risk human papillomavirus (HR-HPV) infection may be contributing factors to oral carcinogenesis at a young age. Therefore, the aim of this study was to evaluate the immunoexpression of cell cycle proteins according HPV status in OSCC affecting young patients.
A tissue microarray construction based on 34 OSCC samples from young patients (<40 years old) was subjected to immunohistochemical reactions for Ki67, cyclin D1, C-ErbB2, p21, Myc, epidermal growth factor receptor, p53, and p16 antibodies.
The clinicopathologic features and the immunoexpression of all tested proteins were similar in both groups. Patients with HPV-related OSSC tended to have better cancer-specific survival (CSS; 39% vs 60% 5-y CSS), and overall survival (OS; 29.2% vs 60% 5-year OS). However, this difference was not statistically significant.
No significant difference exists in the expression of cell cycle proteins studied between HR-HPV DNA–positive and HR-HPV DNA–negative OSCC affecting young patients.
Clinicopathologic and molecular markers in cervical carcinoma: a prospective cohort study
2017, American Journal of Obstetrics and Gynecology
Cervical cancer is a major health problem worldwide. Identification of effective clinicopathologic and molecular markers is vital to improve treatment stratification.
The purpose of this study was to validate a set of well-defined clinicopathologic features in a large population-based, prospectively collected cervical cancer cohort to support their use in the clinic. Further, we explored p53 and human epidermal growth factor receptor 2 as potential prognostic markers in cervical cancer.
Tissue was collected from 401 patients with cervical cancer. Clinical data that included follow-up evaluations were collected from patient journals. Histopathologic data were evaluated and revised by an expert pathologist. The prognostic impact of selected clinicopathologic variables was analyzed in the whole cohort. Tissue microarrays were prepared from 292 carcinomas, and p53 and human epidermal growth factor receptor 2 protein levels were evaluated by immunohistochemistry. Fresh frozen samples from overlapping cervical carcinomas previously were subjected to human papilloma virus typing (n=94), whole exome (n=100) and RNA (n=79) sequencing; the results were available for our analyses.
Among the clinicopathologic variables, vascular space invasion, histologic type, and tumor size were verified as strong independent prognostic markers. High p53 protein levels were associated significantly with markers for aggressive phenotype and survival, also in multivariate survival analysis, but did not reflect TP53 mutational status. High human epidermal growth factor receptor 2 protein levels were identified in 21% of all tumors. ERBB2 amplification was associated with poor outcome (P=.003); human epidermal growth factor receptor 2 protein level was not.
Our findings support that the Féderation Internationale de Gynécologie et d’Obstétrique s guidelines should include vascular space invasion and tumor size 2–4 cm and that careful selection of histologic type is essential for stratification of patient risk groups. High p53 levels independently predict poor survival yet do not reflect mutational status in cervical cancer. Amplified ERBB2 significantly links to poor survival, while HercepTest does not. With optimal stratification, human epidermal growth factor receptor 2–based therapy may improve cervical cancer treatment.
Immunoassays of chemically modified polysaccharides, glycans in glycoproteins and ribose in nucleic acids
2017, Analytica Chimica Acta
Glycosylation of proteins plays an important role in health and diseases. At present new simple and inexpensive methods of glycoprotein analysis are sought. We developed a monoclonal antibody Manost 2.1 in mice after immunization with the adduct of mannan with Os(VI)temed complex (temed is N,N,N′,N'-tetramethylethylenediamine). The specificity of this antibody to different biomolecules treated with Os(VI)temed was tested using dot blot immunoassay. Manost 2.1 showed specificity toward Os(VI)temed-modified polysaccharides, glycoproteins and ribonucleotide at the 3’-end in DNA. The antibody recognized neither the unmodified compounds nor the non-glycosylated proteins treated with Os(VI)temed. We also performed western blotting and Coomassie silver blue staining of mixtures of biomacromolecules treated with Os(VI)temed and identified specifically the modified glycoproteins. The immunochemical method using Manost 2.1 was compared with electrochemical analyses based on redox signals of the Os(VI)temed adducts, with similar results in terms of sensitivity. This new antibody-based approach opens the door for rapid and inexpensive analysis of glycans and glycoproteins in various scientific and medical fields, including cancer research and the future application of glycoprotein detection in clinical practice.

View all citing articles on Scopus

View full text

Research reportAn immunochemical analysis of the human nuclear phosphoprotein p53: New monoclonal antibodies and epitope mapping using recombinant p53

Abstract

Cell

Lancet

Lancet

Virology

Suppression of human colorectal carcinoma cell growth by wild-type p53

Science

Isolation of human p53 specific monoclonal antibodies and their use in the studies of human p53 expression

Eur. J. Biochem.

Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines

Oncogene

Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies

Oncogene

Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer

Oncogene

p53 expression in breast cancer

Int. J. Cancer

Genetic mechanism of tumor suppression by the human p53 gene

Science

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form

EMBO J.

Research report
An immunochemical analysis of the human nuclear phosphoprotein p53: New monoclonal antibodies and epitope mapping using recombinant p53