Inhibition of urinary bladder cancer cell proliferation by silibinin

Silibinin, a natural compound extracted from milk thistle, has demonstrated antitumor properties in urinary bladder cancer cells; however, the role of TP53 gene in these effects is unclear. In order to better understand the molecular and antiproliferative mechanisms of this compound, urinary bladder cancer cells with different TP53 gene status, RT4 (low-grade tumor, wild TP53 gene), 5637 (high-grade tumor, Grade 2, mutated TP53 gene), and T24 (high-grade tumor, Grade 3, mutated TP53 gene) were treated with several concentrations of silibinin (1, 5, 10, 50, 100, and 150 μM). Cytotoxicity, prooxidant effect, morphological changes, cell migration, cell cycle progression, global methylation profile, and relative expression of HOXB3, c-MYC, PLK1, SMAD4, SRC, HAT, HDAC, and RASSF1A genes were evaluated. The silibinin presented cytotoxic and prooxidant effects in the three cell lines. In mutated TP53 cells, significant interference in cell migration and cell cycle arrest at the G2/M phase was observed. Additionally, silibinin induced global DNA hypomethylation in the highest grade tumor cells. For wild-type TP53 cells, a sub-G1 apoptotic population was present. Furthermore, there was modulation of gene expression responsible for cell growth (SMAD and c-MYC), migration (SRC), cell cycle kinetics (PLK1), angiogenesis (HOXB3), and of genes associated with epigenetic events such as DNA acetylation (HAT) and deacetylation (HDAC). In conclusion, the silibinin inhibited the urinary bladder tumor cell proliferation independently of TP53 status; however, cell cycle effects, gene expression changes, and alteration of cell migration are dependent on TP53 status. © 2020 Wiley Periodicals, Inc.     

Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin‐remodelling genes as major players and a prognostic role for TERT,RB1, MEN1 and KMT2D

Next‐generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large‐cell neuroendocrine carcinomas, and 33 small‐cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole‐exome sequencing and high‐coverage targeted sequencing of 418 genes. Eighty‐eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin‐remodelling genes, including those encoding histone modifiers and members of SWI–SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The effect of different TP53 mutations on the chromosomal stability of a human colonic adenoma derived cell line with endogenous wild type TP53 activity,before and after DNA damage

We examined the effect of loss of wild type TP53 activity on the chromosomal stability of a human colonic adenoma derived cell line (designated AA/C1) by studying transfected variants which express different TP53 mutations. Using gross chromosomal aberrations as a measure of instability, we studied metaphase spreads of a vector control cell line (AA/PCMV) and variants expressing the 143(Val-Ala) mutation, which retain endogenous wild type TP53 activity, or the 273(Arg-His) TP53 mutation, which acts as a dominant negative. It was found that the proportion of cells with more than 4% aberrations was significantly greater in the AA/273p53/B cell line (an approximate 5-Fold increase) than in the vector control or the AA/143p53/A cell line. To investigate whether loss of TP53 dependent checkpoints also predisposed the cells to accumulate persistent chromosomal aberrations after DNA damage, cells were exposed to 5 Gy γ radiation. Regardless of TP53 status, cells with radiation induced chromosomal damage were eliminated through a TP53 independent mechanism, suggesting that loss of TP53 activity did not permit the survival of these cells. In contrast, when exposed to low level γ radiation (0.2 Gy), decreased wild type TP53 function and/or expression of mutant TP53 protein led to increased radioresistance (both in the non-dominant as well as the dominant mutant expressing cell lines). These findings suggest that loss of TP53 activity and/or acquisition of specific TP53 mutations can increase chromosomal instability and resistance to low level DNA damage in human colonic adenoma cells. This study emphasises the different biological consequences of individual TP53 mutations on the genotype of premalignant colorectal epithelial cells and subsequent implications for tumorigenic progression. Genes Chromosom. Cancer 20:44–52, 1997. © 1997 Wiley-Liss, Inc.     

Impact of p53 function on the sulfotransferase-mediated bioactivation of the alkylated polycyclic aromatic hydrocarbon 1-hydroxymethylpyrene in vitro

The tumor suppressor p53, encoded by TP53, is known as the “guardian of the genome.” Sulfotransferases (SULTs) are involved in the metabolism of alkylated polycyclic aromatic hydrocarbons such as 1-hydroxymethylpyrene (1-HMP), which is a known substrate for SULT1A1. To investigate the impact of TP53 on the metabolic activation of 1-HMP, a panel of isogenic human colorectal HCT116 cells having TP53(+/+), TP53(+/−), or TP53(−/−) were treated with 10 μM 1-HMP for 24 hr. 1-HMP-DNA adduct formation was determined by ultraperformance liquid chromatography-tandem mass spectrometry analysis, which quantified two nucleoside adducts N²-(1-methylpyrenyl)-2′-deoxyguanosine and N⁶-(1-methylpyrenyl)-2′-deoxyadenosine. 1-HMP treatment resulted in significantly (~40-fold) higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. Higher levels of 1-HMP-induced DNA adducts in TP53(+/+) cells correlated with higher basal expression of SULT1A1/3 in this cell line, but 1-HMP treatment showed no effect on the expression of this protein. These results indicate that the cellular TP53 status is linked to the SULT1A1/3-mediated bioactivation of 1-HMP, thereby broadening the spectrum of p53's targets. Environ. Mol. Mutagen., 60:752–758, 2019. © 2019 Wiley Periodicals, Inc.     

Inactivation of RB1, CDKN2A,and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.     

The spectrum of TP53 mutations in bladder carcinoma

The mutational spectrum for the TP53 gene was investigated in a large series of bladder tumors and bladder tumor cell lines. Tumors and cell lines were screened for the presence of TP53 point mutations by single-strand conformational polymorphism analysis followed by direct sequencing. Mutations were detected in 16 of 88 (18%) tumors and 4 of 14 cell lines (28%). In total, twelve missense mutations, one nonsense mutation, three deletions, and two insertions were identified by direct sequencing. Of the thirteen point mutations sequenced, only one was a transition at a CpG site, whereas five G:C → T:A transversions were found, suggesting a major role for exogenous mutagens in bladder tumorigenesis. Tumors were also examined for loss of heterozygosity (LOH) on chromosome arm 17p. LOH of one or more markers on 17p was detected in 31 % of tumors. All eight tumors with a TP53 mutation from patients informative at TP53 had LOH, whereas nine tumors with LOH at TP53 did not have an identified mutation. Three tumors had LOH on 17p at sites distal to the TP53 locus but retained both TP53 alleles, suggesting the involvement of another tumor suppressor gene on 17p in bladder tumorigenesis in some tumors. Genes Chrom Cancer 9:108-118 (1994).© 1994 Wiley-Liss, Inc.     

Hepatocellular cancer cell lines,Hep-3B and Hep-G2 display the pleiotropic response to resveratrol and berberine

PurposeHuman carcinoma cells with different p53 status exposed to a combination of bioactive substances, resveratrol and berberine, revealed different responses in cell viability via p53-dependant apoptosis pathway activation.Materials and methodsUsing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, we investigated various and opposing effects in hepatocellular carcinoma cells, Hep-G2 and Hep-3B with different p53-status.ResultsCells decreased in viability after treatment with dose-dependent concentrations of resveratrol and berberine. Hep-3B p53 mutants were more sensitive in comparison to the p53 wild type Hep-G2 cell line. A synergistic effect was observed after treatment of Hep-3B cells with a combination of resveratrol/berberine ratios in favor of resveratrol (2:1, 3:1). The results suggest that an effective concentration of berberine, in the presence of resveratrol, could be decreased even to 50% (half the IC₅₀ for berberine) in cancer treatment. Combined treatment with berberine and resveratrol, at the investigated concentrations and fractions, significantly reduces the viability of wild type p53 Hep-G2 and null p53-mutant Hep-3B cells by 20% and 40%, respectively.ConclusionsStronger toxic effects on viability and proliferation were observed in Hep-3B cells what is consistent with the assumptions that null p53-mutants activate apoptosis canonical pathway. In conclusion, p53 status in human hepatocellular cancer cell lines modulates responses to plant-derived therapies.     

Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumor development

As tumor protein 53 (p53) isoforms have tumor‐promoting, migration, and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full‐length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT‐qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumor‐associated macrophage content, and various immune cell markers. Elevated levels of Δ133p53β mRNA characterised glioblastomas with increased CD163‐positive macrophages and wild‐type TP53. In situ‐based analyses found Δ133p53β expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C‐C motif chemokine ligand 2 (CCL2) expressed. Tumors with increased Δ133p53β had increased numbers of cells positive for macrophage colony‐stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine ‘mimic’ of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest that elevated Δ133p53β is an alternative pathway to TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53β to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Mutant p53 accumulation in human breast cancer is not an intrinsic property or dependent on structural or functional disruption but is regulated by exogenous stress and receptor status

Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild‐type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure–function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild‐type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor‐positive or HER2‐positive breast cancers and cell lines than in triple‐negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild‐type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Different TP53 mutations are associated with specific chromosomal rearrangements,telomere length changes,and remodeling of the nuclear architecture of telomeres

TP53 mutations are the most common mutations in human cancers, and TP53‐R175H and TP53‐R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53‐V143A, TP53‐R175H, and TP53‐R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three‐dimensional (3D) quantitative FISH (Q‐FISH) on interphase and Q‐FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53‐R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53‐R175H and TP53‐V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53‐R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53‐R175H. Taken together, our results indicate that the mutation TP53‐R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major determinants of the TP53‐dependent genomic instability. © 2014 Wiley Periodicals, Inc.     

Genetic instability and recurrent MYC amplification in ALK‐translocated NSCLC: a central role of TP53 mutations

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non‐small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co‐occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53‐mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC‐overexpressing ALK+ TP53‐mutated cells had a proliferative advantage compared to wild‐type cells. ChIP‐Seq data revealed MYC‐binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53‐mutated cells resulted in an upregulation of EML4–ALK, indicating a potential MYC‐dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co‐occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The percentage of cells with 17p deletion and the size of 17p deletion subclones show prognostic significance in chronic lymphocytic leukemia

TP53 disruption is considered to be the most important prognostic factor in chronic lymphocytic leukemia (CLL), but not all patients with TP53 disruption have similar dismal outcomes. We evaluated the prognostic value of TP53 disruption in CLL patients without treatment indications. Data of 305 CLL patients were analyzed. 41 of them (13%) had TP53 disruption. Patients with lower percentage of cells with del(17p) had significantly better survival. Patients with mutated IGHV, β2‐microglobulin ≤3.5 mg/L, wild‐type TP53, age ≤65 years or without complex karyotype (CK) had relatively favorable outcomes in the del(17p) group. Furthermore, patients with del(17p) as a minor clone showed survival advantage compared with those with del(17p) as a major clone. These data suggest that the percentage of cells with del(17p), the size of the del(17p) subclone, CLL International Prognostic Index, and CK should be considered to build refined prognostication models for patients with TP53 disruption.     

Molecular alterations in the TP53 gene of peripheral blood cells of patients with chronic myeloid leukemia

The TP53 gene has been extensively studied in patients with chronic myeloid leukemia (CML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. We report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic DNA, we sequenced PCR products of the coding exons and most introns of the TP53 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP53 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP53 mutations, we believe that wild type TP53 accumulates in blood cells of CML patients. Our results, therefore, indicate that molecular changes in coding regions of the TP53 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP53 in CML cells may indicate an additional mechanism involving this gene in the pathogenesis of this disease. Genes Chromosomes Cancer 21:2–7, 1998. © 1998 Wiley-Liss, Inc.     

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells

The mechanisms by which arsenic‐induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT‐PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose‐dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome‐wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long‐lasting arsenic‐induced genomic instability that manifests in several ways, including aberrant chromosomal effects. Environ. Mol. Mutagen. 57:137–150, 2016. © 2015 Wiley Periodicals, Inc.     

Use of multivariate analysis to suggest a new molecular classification of colorectal cancer

Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53–CIN–MSI–BRAF–KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI⁺ and/or BRAF‐mutant; (2) CIN⁺ and/or TP53^– mutant, with wild‐type KRAS and PIK3CA; (3) KRAS‐ and/or PIK3CA‐mutant, CIN⁺, TP53‐wild‐type; (4) KRAS^– and/or PIK3CA‐mutant, CIN^–, TP53‐wild‐type; (5) NRAS‐mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN⁺ CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN⁺ cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease‐free survival. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock‐in (Hupki) mouse embryo fibroblasts

Approximately 50% of human tumors have a mutation in TP53. The pattern and spectra of TP53 mutations often differ between cancer types, perhaps due to different etiological factors. The Hupki (human TP53 knock‐in) mouse embryo fibroblast (HUF) immortalization assay is useful for studying mutagenesis in the human TP53 gene by environmental carcinogens. Prior to initiating an immortalization assay, carcinogen treatment conditions must be optimized, which can require a large number of cells. As primary HUF cultures senesce within 2 weeks, restricting their use, we investigated whether immortalized HUFs retaining wild‐type TP53 can be surrogates for primary HUFs in initial treatment optimization. DNA damage by eight compounds found in diesel exhaust, benzo[a]pyrene, 3‐nitrobenzanthrone, 1‐nitropyrene, 1,3‐dinitropyrene, 1,6‐dinitropyrene, 1,8‐dinitropyrene, 6‐nitrochrysene, and 3‐nitrofluorene, was assessed by ³²P‐postlabeling and the alkaline comet assay in primary HUFs and in an immortal HUF cell line J201. For most compounds, higher levels of DNA adducts accumulated in J201 cells than in primary HUFs. This difference was not reflected in the comet assay or by cell viability changes. Experiments in three additional immortal HUF cell lines (AAI49, U56, and E2‐143) confirmed strong differences in DNA adduct levels compared with primary HUFs. However, these did not correlate with the protein expression of Nqo1 or Nat1/2, or with gene expression of Cyp1a1 or Cyp1b1. Our results show that using immortal HUFs as surrogates for primary HUFs in genotoxicity screening has limitations and that DNA adduct formation is the best measure of genotoxicity of the nitro‐polycyclic aromatic hydrocarbons tested in HUFs. Environ. Mol. Mutagen. 2012. © 2011 Wiley Periodicals, Inc.     

Isolation of a novel TP53 target gene from a colon cancer cell line carrying a highly regulated wild-type TP53 expression system

We established a colon cancer cell line SW480-LOWTP53-1 carrying a wild-type TP53 transgene that is inducible under control of the lactose operon. Induction of this transgene by isopropyl-β-D-thiogalactoside (IPTG) arrests growth of the transfected cells. To investigate cellular responses related to the TP53 signaling pathway to induce growth arrest, we applied a differential display method to screen mRNAs isolated from this cell line and looked for genes whose expression was activated or suppressed after induction of wild-type TP53. Subsequent Northern blot analysis confirmed that expression of one novel gene was regulated by wild-type TP53. The cDNA, termed TP53TG1 (TP53 target gene 1), contained an open reading frame of 270 nucleotides encoding 90 amino acids. Under conditions of cellular stress (ultraviolet irradiation or exposure to bleomycin or cisplatin), expression of TP53TG1 was induced in a wild-type TP53-dependent manner, indicating that this gene is likely to play an important role in the signaling pathway of TP53 and may function in response to cellular damage. Genes Chromosomes Cancer 23:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Recurrent missense variants in clonal hematopoiesis-related genes present in the general population

Clonal hematopoiesis (CH) consists in an abnormal expansion of a hematopoietic stem cell bearing an advantageous somatic variant. A survey of known recurrent somatic missense variants in DNMT3A, SF3B1, SRSF2, and TP53, some of the most prominent genes underlying CH of indeterminate potential (CHIP), in gnomAD noncancer database shows the presence of 73 variants. Many of them reach frequencies higher than 0.01% in various populations and, in many cases, are enriched in specific populations. Consistent with a potential involvement in CHIP, we found that the age distribution of the carriers is shifted towards old ages. Moreover, the variant allele frequencies are on average lower than 50%, expected for germline heterozygous variants. The pervasive presence of some of such variants in blood DNA from elder individuals is compatible with CHIP of somatic origin. On practical grounds, CHIP can lead to misclassification of somatic variants in cancer-predisposition genes as inherited, which bear consequences for the affected individuals and their families.