Expressed P53 Protein Research Articles

Abstract LB180: SPOC, A highly multiplexed platform to study the kinetics of hundreds of biomolecular interactions: applications for early cancer detection

Abstract Tissue specific tumor neo-antigens and tumor associated antigens (TAAs) play a pivotal role in immune system interaction with cancer cells. Mutated, alternately spliced, over-expressed, mis-folded and abnormally post-translationally modified proteins may elicit immune responses that suppress tumor development. As these cancer specific proteins are distinct from normal self-proteins they offer the possibility of serving as highly-specific cancer biomarkers for early detection. TAAs are increasingly being targeted for priming the immune response to immunotherapies and cancer vaccines. Consequently, there has been an exponential increase in research on neo-antigen profiling using sequencing and multi-omic technologies. However, proteomic tools capable of studying and profiling the cancer proteome are limited. To contribute to this, we are developing a novel proteomic screening platform termed Sensor-integrated Proteome on Chip (SPOC) that enables high-throughput (HTP) screening of cancer proteins for loss or gain of function analysis, and humoral immune response profiling. SPOC platform enables use of surface plasmon resonance (SPR) biosensing for kinetic interaction profiling of 100s to 1000s of proteins at once, which can be applied to discovery and characterization of autoantibodies and neoantigens. SPOC uses human cell-free in vitro transcription and translation to express fusion capture-tagged proteins of interest from DNA plasmids printed in nanowells etched into a silicon slide. As proteins are expressed, they are captured covalently onto an overlaid gold-coated surface in individual spots via the capture ligand. The capture ligand requires the tag to be properly folded for binding, thus proteins on the surface are presumed to be fully folded when captured. When SPOC chips are analyzed using SPR, kinetic measurements of protein interactions can be obtained for each protein spot over time (RMax, Kon, Koff, etc.). SPOC arrays may be probed with a variety of biological molecules including small molecule drugs, antibodies, and other proteins. Data can be analyzed using bioinformatics and machine learning for neo-antigen discovery and to generate distinct signatures of disease. To demonstrate SPOC utility for neo-antigen discovery and characterization, a serum sample with known anti-p53 autoantibodies was analyzed on a SPOC SPR chip containing expressed p53 protein spots distributed throughout the array. Increases in SPR signals were observed for each p53 spot on the array as anticipated. Citation Format: Rebecca Cook, Chidozie Victor Agu, William Martelly, Peter A. Bell, Mukilan Mohan, Bharath Takulapalli. SPOC, A highly multiplexed platform to study the kinetics of hundreds of biomolecular interactions: applications for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB180.

Role of p53 Expression in the Progression of Gastric Cancer in the Indian Population

Introduction: Gastric carcinogenesis frequently affects tumor suppressor TP53 gene, resulting in increased expression of p53 protein and mutations towards later stages of Gastric Carcinoma. Objectives: To analyze p53 protein expression in different stages of gastric cancer to determine if it could be used as a biomarker to allow targeted screening of high risk groups with intestinal metaplasia. Methods: We retrieved 101 formalin fixed paraffin embedded tissue blocks of histopathology proven Gastric Carcinoma specimens and conducted an immunohistochemical study on a subset of 25 cases. Tumor sections were taken with adjacent normal mucosa. p53 expression was evaluated using immunohistochemical markers. With the observations from the study, analysis of p53 expression in different areas of the mucosa was done. Result: Thirteen carcinoma cases (52%) expressed p53 protein with a majority of eight (66.7%) cases showing intensity 3. Normal and metaplastic mucosa did not express p53 protein. In dysplastic mucosa, seven (28%) cases showed p53 expression with intensity ranging from 1 to 3. On comparing the data after excluding the p53 negative regions of normal and metaplastic mucosa, there was no difference with statistical significance. Conclusion: p53 expression is negative in normal and metaplastic gastric mucosa but shows expression in dysplastic and cancer cells. Thus, TP53 mutation is most likely an event towards later stages of carcinogenesis. We are dubious on the ability of p53 to be used as an authentic screening tool at the metaplastic stage to predict progression to carcinoma.

Two Mutations in TP53 Promotes Migration, Invasion, and Proliferation of Human Ovarian Cancer A2780 Cells

The ovarian cancer cell line A2780 without endogenous TP53 expression was selected. A plasmid carrying wild-type TP53, TP53 with two point mutations G199X and V157fs, or an empty vector was transfected in A2780 cells with lipofection. Western blot analysis was used to investigate whether the mutant plasmid affects the expression of P53 protein. In addition, the effect of mutation on the migration of A2780 cells was detected with scratch assay, while that on cell invasion was evaluated with transwell assay. We also studied the effect of mutation on the proliferation of A2780 cells with the cell counting kit-8 (CCK-8) assay. As a result, we found that the cells transfected with TP53 G199X and V157fs mutants and empty vector failed to express P53 protein. The migration ability of the cells expressing the two mutant genes was higher than that of the cells expressing the wild-type gene. The invasion ability of the two mutant groups was significantly higher than that of the wild-type group. Cell proliferation was also higher for the two mutant groups and the empty vector group than for the wild-type group. Thus, the two point mutations in TP53 (G199X and V157fs) gene could terminate the expression of P53 protein and promote migration, invasion, and proliferation of ovarian cancer A2780 cells.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

The Tumor Suppressor p53 in Mucosal Melanoma of the Head and Neck.

Despite worldwide prevention programs, the incidence for cutaneous melanoma is continuously increasing. Mucosal melanoma (MM) represents a rare but highly aggressive phenotype of common melanoma with predilection in the sinonasal system. Far away from ultraviolet sun exposure, the molecular mechanisms underlying tumorigenesis and the highly aggressive clinical behavior are poorly understood. In many solid malignomas of the head and neck region, p53 tumor suppressor functions as oncogene due to p53 protein stabilizing mutation. Interestingly, the vast majority of MM demonstrates constitutively expressed p53 protein, with protein stabilizing mutations being rare. Abrogated activation of p53 target genes results in derogation of the apoptotic signal cascade and contributes to the strong resistance against chemotherapeutic agents activating p53 dependent apoptosis. The current review illustrates the role of p53 and its pathway in MM.

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System.

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.

0404 inhibits hepatocellular carcinoma through a p53/miR-34a/SIRT1 positive feedback loop

DNA-damaging agents have been used in cancer chemotherapy for a long history. Unfortunately, chemotherapeutic treatment strategies against hepatocellular carcinoma (HCC) are still ineffective. We screened a novel DNA-damaging compound, designated as 0404, by using time-dependent cellular response profiling (TCRP) based on unique DNA-damage characteristics. We used human HCC cell lines and HCC xenograft mouse model to analyze the anti-cancer effects of 0404. Transcriptome and miRNA arrays were used to verify the anti-cancer mechanism of 0404. It was confirmed that p53 signaling pathway was crucial in 0404 anti-cancer activity and the expression of miR-34a, a key tumor-suppressive miRNA, was up-regulated in 0404-treated HepG2 cells. MiR-34a expression was also down-regulated in HCCs compared with corresponding non-cancerous hepatic tissues. We further identified the mechanisms of 0404 in HepG2 cells. 0404 increased miR-34a expression and acylation p53 protein levels and decreased SIRT1 protein levels in a concentration-dependent manner. The sensitivity of HepG2 cells to 0404 was significantly decreased by transfection with miR-34a inhibitors and SIRT1 protein levels were up-regulated by miR-34a inhibition. Our findings show that 0404 is probably an attractive agent for treating HCC, especially in HCC with wide type (WT) p53, through forming a p53/miR-34a/SIRT1 signal feedback loop to promote cell apoptosis.

Human papillomavirus 16 is an aetiological factor of scrotal cancer

Background:Squamous cell scrotal carcinoma (SCSC) is an infrequent skin cancer associated historically with occupational carcinogens. Human papillomavirus (HPV) DNA has been associated with SCSC but there is no definitive proof of its oncogenic role.Methods:Human papillomavirus-DNA and –E6*I mRNA were analysed in six invasive histologically typed SCSC. LCM-PCR was used to localise HPV DNA to tumour cells. P16INK4aand p53 expression were studied by immunohistochemistry.Results:In three warty or basaloid SCSC HPV16-DNA and E6*I-mRNA were detected. LCM-PCR confirmed HPV16 was in p16INK4a-positive malignant cells. However, of three usual-type SCSC, all were HPV-negative and two expressed p53 protein but not p16INK4a.Conclusions:Human papillomavirus 16 was present in tumour cells and oncogenically active in basaloid and warty SCSC, whereas usual SCSC was HPV-negative and showed immunostaining, suggesting p53 mutation. The dual pathways of oncogenesis and relation between histological type of SCSC and HPV are similar to that in penile cancers.

The Novel Thiosemicarbazone Derivative Coti-2 Induces Mitochondrial Apoptosis in Acute Myeloid Leukemia Cells Via a p53-Independent Mechanism

IntroductionTP53 mutations are rare (< 5%) in de novo acute myeloid leukemia (AML) but if present, they are associated with a very poor prognosis (< 1% overall survival at 3 years). p53 mutations have been frequently detected in poor-prognosis patients with complex karyotype or therapy-related AML. COTI-2 is a third generation thiosemicarbazone derivative that was identified as a small molecule candidate against a diverse group of human cancer cell lines using a proprietary in silico drug screening method. COTI-2 causes cancer cell death via apoptosis. COTI-2 has been proposed to restore non-functional mutant p53 conformation to functional wild-type conformation in solid cancer cells, and it has recently entered a clinical trial in patients with advanced or recurrent gynecologic malignancies (NCT02433626). The molecular mechanisms of COTI-2 to induce apoptosis, however, remain largely unknown. In this study, we investigated activity and possible modes of action of COTI-2 against AML cells, especially focusing on its p53-independent properties.METHODS AND RESULTS A total of 10 AML cell lines were exposed to COTI-2 for 72 hours and its anti-leukemia effects were determined by viable cell counts and annexin V staining. OCI-AML2, OCI-AML3, MOLM-13, MOLM-14 and MV4;11 express wild-type (WT) p53; THP-1and Kasumi-1 express mutant (MUT) p53; KG-1, U937 and HL-60 does not express p53 protein (p53 NULL). COTI-2 inhibited leukemia cell growth and increased the percentage of annexin V-positive cells, irrespective of cellular p53 status. The IC50 values (concentration at which cell growth is inhibited by 50%) for were not statistically different between p53 WT and MUT/NULL cells (10.3 ± 4.5 nM vs 20.2 ± 11.5 nM, P = 0.44). So did the ED50 values (effective concentration inducing 50% cell killing as measured by Annexin V positivity) (115.0 ± 50.9 nM vs 237.8 ± 109.9 nM, P = 0.34). We next examined the apoptotic effect on primary AML cells from patients with AML. A total of 14 samples [11 p53 WT cases and 3 p53 MUT (C135W, R248Q and C242Y) cases] were examined. COTI-2 induced apoptosis both in p53 WT (40.3 ± 6.4% compound-specific annexin V induction) and MUT samples (48.0 ± 17.7%) (P = 0.62). To elucidate the p53-independent activity of COTI-2, mRNA expression levels of TP53 (p53), CDKN1A (p21) and BBC3 (PUMA) were determined in four AML cell lines (OCI-AML3, MV4;11, HL-60 and Kasumi-1) after exposure to COTI-2. COTI-2 did not induce TP53 or its transcriptional targets CDKN1A or BBC3 in any of the cell lines. To investigate if COTI-2 activates an intrinsic pathway to induce apoptosis in AML, we used p53 NULL HL-60 cells overexpressing BCL-2 (HL-60/BCL-2), BCL-XL (HL-60/BCL-XL) and their controls (HL-60/neo). HL-60/BCL-2 and HL-60/BCL-XL respectively expressed BCL-2 and BCL-XL at greater than 4 times higher levels than HL-60/neo. Interestingly, overexpression of either BCL-2 or BCL-XL almost completely abrogated COTI-2-induced apoptosis. Similar results were obtained in OCI-AML3/BCL-2. MCL-1 overexpression only modestly inhibited COTI-2-induced apoptosis. Involvement of the extrinsic pathway was modest as JurkatI9.2 (a Jurkat clone deficient in caspase-8) and its control showed similar sensitivity to COTI-2. COTI-2 appeared to reduce MCL-1 expression levels through mTORC1 inhibition. Finally, COTI-2 strongly synergized with the BCL-2 inhibitor ABT-199 to induce apoptosis in AML cells.Conclusion COTI-2 induces mitochondrial apoptosis in AML cells, irrespective of their p53 mutational status. COTI-2-induced apoptosis depends on BCL-2 and BCL-XL expression but not on MCL1 expression or p53 activation. COTI-2 has clinical potential in AML that often expresses high levels of MCL-1, especially in combination with BCL-2 inhibition. DisclosuresDanter:Critical Outcomes: Employment.

Polyethyleneimine-modified calcium carbonate nanoparticles for p53 gene delivery.

In this study, calcium carbonate (CaCO3) nanoparticles with spherical structure were regulated by arginine and successfully synthesized via a facile co-precipitation method. The average particle size of as-prepared CaCO3 was about 900 nm. The properties of nanostructured CaCO3 particles were characterized by scanning electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction and size distribution. After modified with polyethyleneimine (PEI), the ability of PEI-CaCO3 nanoparticles to carry GFP-marked p53 gene (pEGFP-C1-p53) into cancer cells to express P53 protein were studied. Meanwhile, the cytotoxicity, transfection efficiency, cells growth inhibition and the ability to induce apoptosis by expressed P53 protein were conducted to evaluate the performances of PEI-CaCO3 nanoparticles. The results show that prepared PEI-CaCO3 nanoparticles had good biocompatibility and low cytotoxicity in a certain concentration range. PEI-CaCO3 effectively transfected pEGFP-C1 gene into epithelial-like cancer cells. And with the expression of GFP-P53 fusion protein, pEGFP-C1-p53-gene-loaded PEI-CaCO3 particles significantly reduced the proliferation of cancer cells. These findings indicate that our PEI-modified CaCO3 nanoparticles are potential to be successfully used as carriers for gene therapy.

MiR-375 is Essential for Human Spinal Motor Neuron Development and May Be Involved in Motor Neuron Degeneration.

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.

CD150(-) Side Population Defines Leukemia Stem Cells in a BALB/c Mouse Model of CML and Is Depleted by Genetic Loss of SIRT1.

Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease, and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here, we show that lineage negative CD150(-) side population (CD150(-)SP), but not CD150(+)SP, are CML LSCs in this model, although both CD150(-)SP and CD150(+)SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study, we demonstrate that SIRT1 homozygous knockout substantially reduces CD150(-)SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150(-)SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through deacetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs.

The effect of Zhangfei/CREBZF on cell growth, differentiation, apoptosis, migration, and the unfolded protein response in several canine osteosarcoma cell lines.

BackgroundWe had previously shown that the bLZip domain-containing transcription factor, Zhangfei/CREBZF inhibits the growth and the unfolded protein response (UPR) in cells of the D–17 canine osteosarcoma (OS) line and that the effects of Zhangfei are mediated by it stabilizing the tumour suppressor protein p53. To determine if our observations with D-17 cells applied more universally to canine OS, we examined three other independently isolated canine OS cell lines—Abrams, McKinley and Gracie.ResultsLike D–17, the three cell lines expressed p53 proteins that were capable of activating promoters with p53 response elements on their own, and synergistically with Zhangfei. Furthermore, as with D–17 cells, Zhangfei suppressed the growth and UPR-related transcripts in the OS cell lines. Zhangfei also induced the activation of osteocalcin expression, a marker of osteoblast differentiation and triggered programmed cell death.ConclusionsOsteosarcomas are common malignancies in large breeds of dogs. Although there has been dramatic progress in their treatment, these therapies often fail, leading to recurrence of the tumour and metastatic spread. Our results indicate that induction of the expression of Zhangfei in OS, where p53 is functional, may be an effective modality for the treatment of OS.

Antimicrobial and antiproliferative prospective of kosinostatin – a secondary metabolite isolated from Streptomyces sp.

Cancer is a communal health hazard worldwide. The present investigation attempts to evaluate antimicrobial and anticancer potential of kosinostatin on mammary carcinoma cell line (MCF-7). The anticancer and antiproliferative activities of kosinostatin were analyzed on MCF cell line by MTT assay and cytotoxicity assays like lactate dehydrogenase (LDH) and glutathione (GSH). The secondary metabolite kosinostatin exhibited its apoptotic nature by expressing p53 protein. Collectively, the results acquired from this study promise that kosinostatin shows the potent anticancer activity.

Copper thiosemicarbazones: Antiproliferative action against C6 glioma cells

2(E)-2-[1-(4-pyridinyl)ethylidene]hydrazinecarbothioammidehydrochloride (1) and two of its copper complexes were synthesized and structurally characterized. Cu(I) polymeric complex {[Cu(SCN)(3-Acpy)2]}n (2) was synthesized by in situ mixing of Cu(BF 4 ) 2. 6H 2 O, 4-acetylpyridine (4-Acpy) and semicarbazide whereas a dinuclear Cu(II) complex (3) was obtained from the reaction of Cu(BF 4 ) 2. 6H 2 O, 4-acetylpyridine and ammonium thiocyanate. Magnetic measurements were performed for the dinuclear complex. All the thiosemicarbazones were cytotoxic against malignant RT2 glioblastoma cells (expressing p53 protein) with IC 50 values in the 5.1-13.2 µM range, and against malignant T98 glioblastoma cells (expressing mutant p53 protein) with IC 50 values in the 4.5-31 µM range. Coordination to copper strongly increased the cytotoxic potential in complexes 2 and 3, when compared to that of free ligand and were found to be more potent than cisplatin. All the test compounds (1-3) were able to induce cell death by apoptosis. DOI: http://dx.doi.org/10.3329/bjp.v9i4.19772

An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas

BackgroundInactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.MethodsWe analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.Results50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.ConclusionsThe results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.

Probing the Hepatic Progenitor Cell in Human Hepatocellular Carcinoma

Objective. The intrahepatic stem cells, also known as hepatic progenitor cells (HPCs), are able to differentiate into hepatocytes and bile duct epithelia. By exposure of different injuries and different hepatocarcinogenic regimens, the mature hepatocytes can no longer effectively regenerate; stem cells are involved in the pathogenesis of hepatocellular carcinoma. Methods. Immunohistochemistry was performed on 107 paraffin-embedded hepatocellular carcinoma specimens with the marker of hepatocyte and hepatocellular carcinoma (HepPar1), biliary differentiation (CK7,CK19), haemopoietic stem cell (HSC) (c-kit/CD117, CD34, and Thy-1/CD90), HPC specific markers (OV-6), and Ki-67, p53 protein. Results. HPCs can be identified in the tumor nodules, around the edge of tumor nodules, and in the portal tracts of the paracirrhosis nodules being positive in HepPar1, CK7, CK19, and OV-6, but they failed to immunostain with CD117, CD34, and CD90. The HPCs positive in Ki-67 are observed in the tumor and paracirrhosis tissues. In 107 specimens, 40.2% (43/107) HCC tissues expressed p53 protein, lower than that of the HPCs around the tumor nodules (46.7%, 50/107) and much higher than that of the HPCs around the paracirrhosis nodules (8.41%, 9/107). Conclusion. Human hepatocellular carcinogenesis may be based on transformation of HPCs, not HSCs, through the formation of the transitional cells (hepatocyte-like cells and bile ductal cells).

Clinical Significance of Mannose-Binding Lectin Expression in Thyroid Carcinoma Tissues

Mannose-binding lectin (MBL) plays an important role in the host defence against pathogens and carcinogenesis. This study aimed to analyze differential expression of MBL protein in thyroid cancer tissues and then to investigate the effects of rhMBL in thyroid cancer cells. Tissue specimens from 45 thyroid carcinoma patients and 45 adenoma patients were recruited for immunohistochemical analysis of MBL expression. Cell viability, apoptosis, RT-PCR and Western blot assays were used to detect changes in tumor cell viability, apoptosis, and gene expression, respectively, after treatment of thyroid cancer cells with rhMBL. MBL was differentially expressed in papillary thyroid carcinoma, adenoma, and the distant normal tissues (0.322 ± 0.008, 0.227 ± 0.003, and 0.113 ± 0.003, respectively, P < 0.05). MBL expression was associated with the advanced disease stage, histological grade, or lymph node metastasis in cancer patients (P < 0.05). Moreover, rhMBL treatment of thyroid cancer cells reduced tumor cell viability but induced apoptosis in a dose- and time-dependent manner. rhMBL treatment also downregulated Bcl2 protein expression in thyroid cancer cells (P < 0.05). In addition, expression p53 protein was increased in thyroid cancer cells after rhMBL treatment (P < 0.05). The data from the current study demonstrate that MBL overexpression is associated with advanced thyroid carcinomas, and rhMBL treatment significantly reduced viability but induced apoptosis of thyroid cancer cell lines. Further studies will clarify whether overexpressed MBL in thyroid cancer tissues is functional.

Exon 1 Disruption Alters Tissue-Specific Expression of Mouse p53 and Results in Selective Development of B Cell Lymphomas

p53 is a tumor suppressor gene mutated in >50% of human cancers, while p53 deficiency in mice results in cancers and accelerated mortality. Thymic T cell lymphoma is the most common malignancy in p53-deficient mice, making it difficult to study the role of p53 in other malignancies. To overcome this limitation, we attempted to generate mice with a reversible p53 knockout (p53rev/rev) by inserting a floxed transcriptional stop into the first exon of p53, anticipating that this would allow tissue-specific Cre-mediated expression of p53. Contrary to expectations, functional p53 protein was expressed in the thymus and multiple other tissues of p53rev/rev mice in the absence of Cre, whereas B cells expressed p53 protein only in the presence of B cell-specific CD19-Cre. In the absence of Cre, 76% of p53rev/rev mice developed splenic marginal zone B cell lymphomas, indicating sensitivity of this B cell subset to transformation caused by p53 deficiency. 5′-RACE identified p53 mRNA transcribed from a novel start site utilized in thymocytes but not normal B cells or B cell lymphomas from p53rev/rev mice. The p53rev/rev mouse thus demonstrates an effect of p53 deficiency in development of splenic marginal zone lymphomas and provides a model for study of p53-deficient human B cell lymphomas.

Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: report from an International DLBCL Rituximab-CHOP Consortium Program Study

AbstractTP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP–treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 507 cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP–treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.