P53 Gene Deletion Research Articles

Abstract Tu094: p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload

Background: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. Methods: We used the Tcf21 (transcription factor 21) MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Results: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21- lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. Conclusions: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Unveiling the impact of glutathione (GSH) and p53 gene deletion on tumor cell metabolism by amino acid and proteomics analysis.

Tumor cell inhibition is a pivotal focus in anti-cancer research, and extensive investigations have been conducted regarding the role of p53. Numerous studies have highlighted its close association with reactive oxygen species (ROS). However, the precise impact of the antioxidant glutathione (GSH) in this context remains inadequately elucidated. Here, we will elucidate the anti-cancer mechanisms mediated by p53 following treatment with GSH. In this study, we employed a p53 gene knockout approach in SW480 colorectal cells and conducted comprehensive analyses of 20 amino acids and proteomics using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). These analyses unveiled profound alterations in amino acids and proteins triggered by GSH treatment, shedding light on novel phenomena and delineating the intricate interplay between GSH and cellular proteins. The deletion of the p53 gene exerts a profound influence on tumor cell proliferation. Moreover, tumor cell proliferation is significantly affected by elevated GSH levels. Importantly, in the absence of the p53 gene, cells exhibit heightened sensitivity to GSH, leading to inhibited cell growth. The combined therapeutic approach involving GSH and p53 gene deletion expedites the demise of tumor cells. It is noteworthy that this treatment leads to a marked decline in amino acid metabolism, particularly affecting the down-regulation of methionine (Met) and phenylalanine (Phe) amino acids. Among the 41 proteins displaying significant changes, 8 exhibit consistent alterations, with 5 experiencing decreased levels and 3 demonstrating increased quantities. These proteins primarily participate in crucial cellular metabolic processes and immune functions. In conclusion, the concurrent administration of GSH treatment and p53 gene deletion triggers substantial modifications in the amino acid and protein metabolism of tumor cells, primarily characterized by down-regulation. This, in turn, compromises cell metabolic activity and immune function, ultimately culminating in the demise of tumor cells. These newfound insights hold promising implications and could pave the way for the development of straightforward and efficacious anti-cancer treatments.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

UroVysion™ fluorescence in situ hybridization (FISH) possibly has a high positive rate in carcinoma of non-urothelial lineages.

Background: Positive UroVysion™ fluorescence in situ hybridization (FISH) is generally considered as urothelial carcinoma (UC). We clarify if UroVysion™ FISH can be positive in carcinoma of non-urothelial lineages (CNUL), and verify the consistency of urine FISH and histological FISH in CNUL. Methods: All CNUL subjects detected by urine FISH assay due to haematuria from Tongji Hospital were screened. Meanwhile, 2 glandular cystitis and 2 urothelial carcinoma were served as negative or positive control. Paraffin-embedded tissue sections of all subjects were sent to the pathology department for histological FISH detection. Results: A total of 27 patients were included in this study, including 9 with adenocarcinomas, 11 with squamous cell carcinomas, and 7 with other tumour types. The overall positive rate in urine FISH was 64.00% (16/25) in patients with CNUL, 77.78% (7/9) in those with adenocarcinoma and 54.55% (6/11) in those with squamous carcinoma. There was a significant difference in the GLP p16 gene deletion rate between UC and CNUL (100% vs. 8.00%, p = 0.017). Histological FISH results showed that the histological results of 19 patients were consistent with their urine FISH results, and only one patient with stage Ⅲa urachal carcinoma had inconsistent histological FISH results (positive) and urine FISH (negative) results. Conclusion: We demonstrated for the first time the application value of FISH in CNUL on urine samples. Positive urine FISH tests indicate not only UC, but also CNUL. UroVysion™ FISH possibly has a high positive rate in CNUL. CNUL and UC have different genetic changes shown by FISH.

Interruption of p53-MDM2 Interaction by Nutlin-3a in Human Lymphoma Cell Models Initiates a Cell-Dependent Global Effect on Transcriptome and Proteome Level.

In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.

P53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Establishment of a novel experimental system using single cell-derived pleomorphic rhabdomyosarcoma cell lines expressing K-RasG12V and deficient in p53.

Pleomorphic rhabdomyosarcoma (PRMS) predominantly arises in adult skeletal musculature and is usually associated with poor prognosis. Thus, effective treatments must be developed. PRMS is a rare tumor; therefore, it is critical to develop an experimental system to understand the cellular and molecular mechanisms of PRMS. We previously demonstrated that PRMS develops after p53 gene deletion and oncogenic K-Ras expression in the skeletal muscle tissue. In that study, oncogenic K-Ras-expressing cells were diverse and the period until disease onset was difficult to control. In this study, we developed an experimental system to address this problem. Single cell-derived murine cell lines, designated as RMS310 and RMSg2, were established by limiting the dilution of cells from a lung metastatic tumor colony that were positive for various cancer stem cells and activated skeletal muscle-resident stem/progenitor cell marker genes by RT-PCR. All cell lines stably recapitulated the histological characteristics of human PRMS as bizarre giant cells, desmin-positive cells, and lung metastases in C57BL/6 mice. All subclones of the RMSg2 cells by the limiting dilution in vitro could seed PRMS subcutaneously, and as few as 500 RMSg2 cells were sufficient to form tumors. These results suggest that the RMSg2 cells are multipotent cancer cells that partially combine the properties of skeletal muscle-resident stem/progenitor cells and high tumorigenicity. Thus, our model system's capacity to regenerate tumor tissue in vivo and maintain stable cells in vitro makes it useful for developing therapeutics to treat PRMS.

Abstract 6034: Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments

Abstract Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common malignancy diagnosed in Caucasians with an incidence of about one million cases per year. TP53 and p16 are the most common mutated genes in cSCC. Previous studies of cSCC have shown that patients with both p53 and p16 mutations have lower survival rates. There are currently no syngeneic mouse preclinical models to accelerate the efficacy of immunotherapy combinations to treat cSCC. Our laboratory has generated several genetically engineered mouse models (GEMMs) containing tissue-specific p53 and p16 gene mutations. Recently, we have immortalized three murine cSCC cell lines (cSCC-5690, cSCC-8006, and cSCC-8271) derived from spontaneous skin tumors from GEMMs. Therefore, we hypothesized that the double mutant cell line cSCC-5690 enhances cell survival, tumor aggressiveness, and has lower sensitivity to antibody immunotherapy. Protein and RNA expression of the cell lines were validated by western blotting and qRT-PCR assays. Sphere and soft agar assays were used to evaluate the stem cell population and tumorigenicity of the cell lines. Then, we examined the chemosensitivity with cisplatin through clonogenic assays. To further study the tumorigenicity of cells, we evaluated in vivo tumor growth on C57BL/6J immunocompetent mice by injecting subcutaneously the tumor cell lines. The three cell lines expressed different levels of keratin 14 (K14), but cSCC 5690 and 8006 expressed high levels of genes related to oncogenic pathways as well as mesenchymal markers. The cell line cSCC-5690 expressing mutant p53 R172H with p16 gene deletion, showed an enhanced sphere, and colony tumor formation and both tumorigenic cell lines (cSCC-5690, 8006) showed different sensitivities to cisplatin treatment. A higher tumor growth rate was observed with cSCC-5690 compared to cSCC-8006, while cSCC-8271 showed no tumor growth. Immunohistochemistry studies demonstrated that both cSCC-5690 and 8006 tumors expressed vimentin but only cSCC-5690 expressed K14. The most aggressive syngeneic tumor model (cSCC-5690) was used to evaluate the anti-tumor efficiency of anti-PD1, anti-CD47 antibodies, and STING-agonist. Preliminary results revealed a significant tumor response only to STING agonist (c-di-GMP) which activates the innate and adaptive immune response. Therefore, we were able to characterize all three cell lines and established a new mouse model of cSCC as a relevant platform for immunotherapy studies. Citation Format: Alanis Enid Rodriguez Rosario, Roberto Rangel, Jeffrey Myers. Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6034.

Histomorphological Features of Ovarian Neoplasms and Expression of p53 and WT1 in Surface Epithelial Tumours: A Cross-sectional Study

Introduction: Ovary is the most common site of neoplastic and non neoplastic lesion, can present in childhood to postmenopausal age group and remain the most lethal of all gynaecological malignancies. Tumour Protein (p53) gene mutations or deletions are most common in ovarian carcinoma. However, Wilms' Tumor gene1 (WT1) and p16 expression are also seen in serous ovarian carcinoma. These Immunohistochemistry (IHC) marker are useful in the differential diagnosis of serous ovarian carcinomas. Aim: To study histomorphology of ovarian neoplasm along with expression of p53 and WT1 in surface epithelial tumours and to assess the prevalence of various ovarian neoplasms in different age groups. Materials and Methods: This cross-sectional study was conducted in Department of Pathology, Sarojini Naidu Medical College, Agra, Uttar Pradesh, India, over a period of two years (from September 2018-September 2020). A total of 78 ovarian specimen received in histology laboratory was studied for gross and microscopic features. The p53 and WT1 IHC expression pattern was studied in surface epithelial tumours. Statistical significance was calculated in relation to p53 and WT1 expression with histological type and grade of tumour using Chi-square test. Results: A total of 78 cases were studied, out of which benign tumours were the most common 40 (51.3%) cases, followed by malignant tumours 32 (41.0%) cases and borderline malignancy 6 (7.7%) cases. The most common benign lesions were mucinous cystadenoma 20 (50%) cases. Serous carcinomas were most common malignant tumours 19 (59.3%) cases followed by Germ Cell Tumours (GCT) 5 (15.6%) cases. All benign and borderline epithelial ovarian tumours were found p53 and WT1 negative. Out of 22 cases of malignant surface epithelial tumour, 14 (63.6%) and 12 (54.4%) were positive for p53 and WT1 respectively and all were serous carcinomas. Conclusion: Ovarian lesions present with wide spectrum of histomorphological features. The p53 and WT1 show different rates of expression and staining pattern in various epithelial ovarian carcinomas. Hence routine gross, proper histological examination and correct IHC interpretation is required for specific diagnosis.

Abstract 2963: A novel uterine leiomyosarcoma mouse model

Abstract leiomyosarcoma is an extremely rare but clinically aggressive uterine cancer arising from the smooth muscle of the uterus. Each year, it is estimated to occur in 6 out of every 1,000,000 women in the United States. The average age at diagnosis is 51 and women with uterine leiomyosarcomas have a poor prognosis. Current therapies for uterine leiomyosarcoma are not very effective and the primary treatment of uterine leiomyosarcoma is surgery. Due to the intrinsic rarity of this disease, prospective randomized clinical trials examining the outcome in individuals with uterine leiomyosarcoma is limited. Moreover, a poor understanding of the pathogenesis of this disease is also a major knowledge gap to develop effective treatment strategies. Thus, a preclinical uterine leiomyosarcoma models that recapitulate the human disease is desperately needed to support clinical trial readiness. Since both p53 mutations and Pten homozygous deletion have been observed in 61% and 64% human uterine leiomyosarcomas respectively [3], we are generating genetic mouse models with Amhr2-Cre Ptenfl/fl p53R245Wfl/fl (homozygous p53 mutation), Amhr2-Cre Ptenfl/fl p53R245Wfl/+ (heterozgyous p53 mutation) and Amhr2-Cre Ptenfl/fl (control) genotypes. Our unpublished preliminary data showed that mouse with homozygous p53 mutation and homozygous deletion of Pten developed metastatic uterine leiomyosarcoma at 24 weeks of age with 100% penetrance. However, mouse with heterozygous p53 mutation and homozygous deletion of Pten did not develop uterine leiomyosarcoma at the age of 24 weeks. From the public available human uterine leiomyosarcoma RNAseq data, 80% of those tumors with p53 mutations had homozygous mutations. Previous mouse model with homozygous deletion of p53 gene and/or BRCA1 with Amhr2-Cre only had uterine leiomyosarcoma developed in approximately 50% of mice around 13 months without metastasis. RNAseq and RPPA functional data of the uterine leiomyosarcomas from our novel mouse models are being generated for comparison with known genomic profiles of human uterine leiomyosarcomas. The RNAseq and RPPA functional proteomic data from comparing uterine tissues from homozygous and heterozygous p53 mutated mice provide insight in the understanding the development of uterine leiomyosarcoma and for future clinical trials. Citation Format: Chun Wai Ng, Kwong-Kwok Wong. A novel uterine leiomyosarcoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2963.

Increased Number of Chromosomal Imbalances and High-Level DNA Amplifications in Mantle Cell Lymphoma Are Associated With Blastoid Variants

AbstractMantle cell lymphomas (MCLs) are characterized by 11q13 chromosomal translocations and cyclin D1 overexpression. The secondary genetic and molecular events involved in the progression of these tumors are not well known. In this study, we have analyzed 45 MCLs (32 typical and 13 blastoid variants) by comparative genomic hybridization (CGH). To identify the possible genes included in the abnormal chromosome regions, selected cases were analyzed for P53, P16INK4a, RB, C-MYC, N-MYC, BCL2, BCL6, CDK4, and BMI-1 gene alterations. The most frequent imbalances detected by CGH were gains of chromosomes 3q (49%), 7p (27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%) and losses of chromosomes 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%), 10p14-p15 (18%), 17p (16%), and 9p (16%). High-level DNA amplifications were identified in 11 different regions of the genome, predominantly in 3q27-q29 (13%), 18q23 (9%), and Xq28 (7%). The CGH analysis allowed the identification of regional consensus areas in most of the frequently involved chromosomes. Chromosome gains (P = .02) and losses (P = .01) and DNA amplifications (P = .015) were significantly higher in blastoid variants. The significant differences between blastoid and typical tumors were gains of 3q, 7p, and 12q, and losses of 17p. CGH losses of 17p correlated with P53 gene deletions and mutations. Similarly, gains of 12q and high-level DNA amplifications of 10p12-p13 were associated with CDK4 and BMI-1 gene amplifications, respectively. One of 2 cases with 8q24 amplification showed C-MYC amplification by Southern blot. Alterations in 2p, 3q, 13, and 18q were not associated with N-MYC, BCL6, RB, orBCL2 alterations, respectively, suggesting that other genes may be the targets of these genetic abnormalities in MCLs. Increased number of gains (0 v 1-4 v >4 gains per case) (P = .002), gains of 3q (P = .02), gains of 12q (P = .03), and losses of 9p (P = .003) were significantly associated with a shorter survival of the patients. These results indicate that an increased number of chromosome imbalances are associated with blastoid variants of MCLs and may have prognostic significance.

Diagnostic value of fluorescence in situ hybridization for detecting p16 gene deletion in malignant mesothelioma

Diagnostic value of fluorescence in situ hybridization for detecting p16 gene deletion in malignant mesothelioma

Evaluation of UroVysion for Urachal Carcinoma Detection.

Background: Patients with hematuria who are positive for urinary fluorescence in situ hybridization (FISH) are generally considered to have urothelial carcinoma. We determined whether UroVysion FISH could be used for the diagnosis of urachal carcinoma.Methods: Seven cases of urachal carcinoma with haematuria subjected to FISH analysis were retrospectively analyzed in our hospital from May 2012 to November 2019. Paraffin-embedded tissue sections from one FISH-positive and one FISH-negative urachal carcinoma were processed in strict accordance with the instructions of the UroVysion kit. Meanwhile, FISH data from the other 414 hematuria patients were collected as controls.Results: All 7 patients with urachal carcinoma were diagnosed with adenocarcinoma. According to Sheldon stage, six patients had stage IIIa and one patient had stage IVb. The sensitivity and specificity of urinary FISH for the diagnosis of urachal carcinoma were 71.43% (5/7) and 94.61% (281/297), respectively. The rates of polysomy for chromosomes 3 and 7 in positive patients were both 100% (5/5), whereas the rate of polysomy for chromosome 17 was 40% (2/5), and the chromosome 9p21 region (p16) gene deletion rate was 20% (1/5). Histological assessment and cytological FISH were consistent for urachal carcinoma. No significant difference was observed in the diagnostic efficacy between urachal carcinoma and urothelial carcinoma (71.43 vs. 87.18%, P = 0.245).Conclusions: Taken together, UroVysion FISH was found to be positive in a high proportion of pathologically confirmed urachal carcinoma of late stage with hematuria. Its chromosomal aberrations may be different from those of urothelial carcinoma, but more studies are needed to clarify their genetic background. Not all tumors showing abnormalities by FISH are urothelial carcinomas.

Association between TP53 gene deletion and protein expression in esophageal squamous cell carcinoma and its prognostic significance.

The aim of the present study was to investigate the association between tumor protein 53 (TP53) gene deletion and protein expression and clinical features in esophageal squamous cell carcinoma (ESCC), and to evaluate the predictive value of these two characteristics in the prognosis of ESCC. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed to detect the expression of p53 protein and gene deletion in ESCC tissue samples from different ethnic groups in Xinjiang, in order to analyze their association with clinicopathological characteristics and patient prognosis, as well as the sensitivity and specificity of the two methods. In addition, the results were further validated by tissue microarray from a different region. The positive rate of p53 protein expression was 54.5% (201/369) in the multi-ethnic group, and was significantly different between sex (P=0.026) and between tumor differentiation groups (P=0.032). FISH demonstrated that the TP53 gene deletion rate was 31.8% (68/214), which was significantly different between different tumor differentiation (P=0.002), lymph node metastasis (P=0.005) and vascular invasion (P<0.001) groups. The survival rate of patients with TP53 gene deletion was significantly lower than those without TP53 gene deletion (P<0.05). The positive rate of p53 protein expression in the tissue microarray was 58.1% (68/117), which was significantly different between the depth of invasion groups (P=0.011). The TP53 gene deletion rate was 47.9% (56/117), which significantly differed according to lymph node metastasis (P=0.003) and TNM stage (P=0.01). In addition, the total concordance rates of the two methods were 60.3 and 64.1%, respectively. There were also significant differences in the positive rate of TP53 gene deletion and protein expression in different stages of ESCC (P<0.05), which increased gradually with the progression of ESCC. The deletion of the TP53 gene in esophageal cancer was associated with poor prognosis and may be an important biomarker for evaluating the prognosis of patients with ESCC. The combination of FISH and IHC methods could significantly improve the detection rate of TP53 gene abnormalities and the accuracy of prognostic assessment of ESCC.

Differential effectiveness of tyrosine kinase inhibitors in 2D/3D culture according to cell differentiation, p53 status and mitochondrial respiration in liver cancer cells

Sorafenib and Regorafenib are the recommended first- and second-line therapies in patients with advanced hepatocellular carcinoma (HCC). Lenvatinib and Cabozantinib have shown non-inferior antitumoral activities compared with the corresponding recommended therapies. The clinical trials have established recommended doses for each treatment that lead different blood concentrations in patients for Sorafenib (10 µM), Regorafenib (1 µM), Lenvatinib (0.1 µM), and Cabozantinib (1 µM). However, very low response rates are observed in patients attributed to intrinsic resistances or upregulation of survival signaling. The aim of the study was the comparative dose–response analysis of the drugs (0–100 µM) in well-differentiated (HepG2, Hep3B, and Huh7), moderately (SNU423), and poorly (SNU449) differentiated liver cancer cells in 2D/3D cultures. Cells harbors wild-type p53 (HepG2), non-sense p53 mutation (Hep3B), inframe p53 gene deletion (SNU423), and p53 point mutation (Huh7 and SNU449). The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose–response analysis in 2D cultures showed Sorafenib and Regorafenib were increasingly effective in reducing cell proliferation, and inducing apoptosis in well-differentiated and expressing wild-type p53 in HCC cells. Lenvatinib and Cabozantinib were particularly effective in moderately to poorly differentiated cells with mutated or lacking p53 that have lower basal oxygen consumption rate (OCR), ATP, and maximal respiration capacity than observed in differentiated HCC cells. Sorafenib and Regorafenib downregulated, and Lenvatinib and Cabozantinib upregulated epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor receptor (c-Met) in HepG2 cells. Conclusions: Sorafenib and Regorafenib were especially active in well-differentiated cells, with wild-type p53 and increased mitochondrial respiration. By contrast, Lenvatinib and Cabozantinib appeared more effective in moderately to poorly differentiated cells with mutated p53 and low mitochondrial respiration. The development of strategies that allow us to deliver increased doses in tumors might potentially enhance the effectiveness of the treatments.

Frequency and Role of CDKN2A Deletion in High-Risk Pituitary Neuroendocrine Tumors.

The underlying mechanisms of aggressive pituitary neuroendocrine tumors (pitNETs) are still unclear. The p16 protein, encoded by the CDKN2A tumor suppressor gene on chromosome 9p21, is commonly reported to be lost in numerous types of cancer. For this reason, this study examined to examine the status of homozygous deletion of CDKN2A in high-risk pitNETs. Thirty-eight high-risk pitNETs (30 male, 8 female) were analyzed for CDKN2A deletion by fluorescent in situ hybridization (FISH). Demographic characteristics such as sex, patient age at operation, and sellar magnetic resonance imaging findings including tumor size and invasion status were recorded. The frequency of CDKN2A homozygous deletion by FISH was 3/38 (7.89%) in the high-risk pitNET group. All of these three cases with CDKN2A homozygous deletion were invasive densely granulated lactotroph tumors (p= 0.000). CDKN2A deletion was not correlated with patient age, sex, cavernous sinus invasion (CSI), and tumor size (p> 0.05). The Ki-67 proliferation index was significantly correlated with CDKN2A homozygous deletion (p= 0.003). The mean Ki-67 proliferation index was 10.7% in pitNETs with CDKN2A homozygous deletion and the Ki-67 proliferation index in the whole study group was 4.1%. CSI was significantly correlated with the morphofunctional tumor types including lactotroph tumor, invasive null cell tumor, and invasive gonadotroph tumor (p= 0.021). These findings suggest a close correlation between inactivation of p16 gene and invasive lactotroph tumors. Further investigations are needed to expand on the mechanism of p16 (CDKN2A) gene deletion in high-risk pitNETs.

Fast enzyme-linked electrochemical sensing of DNA hybridization at pencil graphite electrodes. Application to detect gene deletion in a human cell culture

In this paper we present a rapid electrochemical enzyme-linked DNA hybridization assay using disposable pencil graphite electrodes (PeGE) to detect target DNA (tDNA) sequences in DNA fragments amplified by polymerase chain reaction. The procedure consists of several short (1–2 min) incubation steps, including adsorption of the tDNA at unpretreated PeGE from denaturing medium, surface blocking with milk proteins, hybridization with a biotinylated oligonucleotide probe and binding of streptavidin-alkaline phosphatase conjugate to the biotin tags. Then the PeGE is transferred into background electrolyte solution containing 1-naphthyl phosphate, which is enzymatically dephosphorylated to give electrochemically oxidizable indicator 1-naphthol. The assay, which can be performed within 7–8 min, offers a perfect discrimination between specific and nonspecific DNA amplicons and easy detection of about ~40 femtomoles of tDNA in large excesses of non-complementary DNA. An application on the detection of p53 gene deletion in a human cell culture, featuring a real biological model, is presented. The designed setup has a potential to be applied as one of simple, fast, robust ultralow cost “do-it-yourself “instruments recently introduced as diagnostic tools for third world countries.

The Establishment of Esophageal Precancerous Lesion Model by Using p53 Conditional Knockout Mouse in Esophageal Epithelium.

Understanding the molecular mechanisms of precancerous lesion of esophageal cancer is beneficial for early diagnosis and early treatment. The deletion of p53 gene is common in esophageal cancer, but its pathogenesis is still unclear. An animal model is urgently needed to study the mechanisms of esophageal cancer and p53 deficiency. KO mice (p53flox/flox.ED-L2-Cre+/−) and the corresponding control Loxp mice (p53flox/flox.ED-L2-Cre−/−) were obtained by crossing between the p53flox/flox mice and ED-L2-Cre+/− mice. Methylbenzylnitrosamine (NMBA) was injected subcutaneously to induce esophageal precancerous lesion of these two groups of mice. Hematoxylin and eosin staining analysis was performed to evaluate the number and extent of esophageal precancerous lesions in KO mice and Loxp mice at the 16th and 48th weeks. Immunohistochemistry analysis was used to detect the change of Ki67, P21, Bcl-2, and Bax proteins. The number and extent of esophageal precancerous lesions in KO mice were significantly increased compared with the control at the 16th and 48th weeks under the induction of NMBA. The Ki67, P21, Bcl-2, and Bax proteins also had cancer-related pathological characteristics. These results suggest that the esophageal precancerous lesion model was established under the combined effect of p53 gene deletion in esophageal epithelium and NMBA, which could provide a new esophageal precancerous lesion model to explore the mechanism of precancerous lesions.

Cisplatin effect on head and neck squamous cell carcinoma cells is modulated by ERK1/2 protein kinases.

The extracellular signal-regulated kinases (ERKs) are key transducers of the extracellular signals into intracellular responses and represent major molecular players in tumorigenesis. The aim of this study was to determine how curcumin (CRM) used as an adjuvant supports the apoptotic process induced by a single chemical agent treatment (cisplatin-CisPT) on two head and neck squamous cell carcinoma cell lines (FaDu and PE/CA-PJ49) and the involvement of ERK1/2 and/or p53 activation in this process. Data have shown that the CisPt effect is potentiated by CRM. CRM induced an increase of p53 protein phosphorylation in both cell lines. CisPt decreased p53 protein phosphorylation in FaDu cells, but increased it in PE/CA-PJ49 cells. Data showed that the constitutive expression of activated ERK1/2 protein-kinase was different in the two analyzed tumor cell lines. ERK1/2 activation status was essential for both cell processes, proliferation and apoptosis induced by CisPt and/or CRM treatment on squamous cell carcinoma cells. Our data suggest that p53 phosphorylation in the apoptotic process induced by CRM treatment might require the involvement of ERK1/2. In this regard the CisPt treatment suggested that p53 phosphorylation is ERK1/2 independent in FaDu cells having a p53 gene deletion and ERK1/2 dependent in PE/CA-PJ49 cells having a p53 gene amplification. Moreover, in both tumor cell lines our results support the involvement of p53 phosphorylation-ERK1/2 activation-dependent in the apoptosis induced by combined treatments (CisPt and CRM). The use of CRM as adjuvant could increase the efficiency of chemotherapy by modulating cellular activation processes of ERK1/2 signaling pathways. In conclusion, the particular mode of intervention by which ERK1/2 might influence cell proliferation and/or apoptosis processes depends on the type of therapeutic agent, the cells' particularities, and the activation status of the ERK1/2.

Clinical feature and cytogenetic analysis of 28 patients with bone marrow invasion non-Hodgkin's lymphoma

To study the correlation of hematomorphology, bone marrow cytogenetics and clinical biochemical parameters with the prognosis of non-Hodgkin's lymphoma with bone marrow invasion. Morphological analysis of bone marrow cells was performed by routine bone marrow puncture.Chromosome samples were prepared by short-term bone marrow culture. Karyotype analysis was carried out by R-banding in 28 patients. P53 gene was detected by fluorescence in situ hybridization (FISH). Serum lactate dehydrogenase (LDH) of all patients was determined and compared. In all patients, bone marrow morphology showed invasion of lymphoma. Chromosome analysis revealed abnormal karyotypes in 19 cases, which yielded an incidence of 67.85%. The proportion of lymphoma cells in bone marrow among those with an abnormal karyotype was much higher than those with a normal karyotype (60.2% vs. 33.5%, P<0.05). FISH assay showed that 9 (32.14%) patients had P53 gene deletion. And the deletion was much more common among those with an abnormal karyotype (42.11% vs. 11.11%, P<0.05). The serum LDH level in patients with an abnormal karyotype was significantly higher compared with whose with a normal karyotype (1464.37 U/L vs. 294.33 U/L, P<0.05). Patients with abnormal karyotypes have a higher rate of P53 gene deletion, and their LDH level is significantly higher than those with a normal karyotype, which predicted a relatively poor prognosis.