Different mutational characteristics of TSG in cell lines and surgical specimens.

One of the most crucial concerns of cancer research pertains to the differences between the neoplastic cells in tumor specimens in vivo and their counterparts in cell lines. The huge amount of results deposited in cancer genetic databases allows to address this issue from a wider perspective. Our analysis of the Sanger Institute Catalog Of Somatic Mutations In Cancer (COSMIC) database v61 showed a lower percentage of homozygous mutations in a group of tumor suppressor genes in surgical samples (in vivo) in comparison to their frequency in cell lines (in vitro). Similarly, the mutations resulting in the lack of protein (e.g., nonsense mutations or whole gene deletions) of several tumor suppressor genes (TSGs) were more frequently observed in vitro than in vivo. In this article, we suggest two potential explanations of these data. Firstly, TSG heterozygous mutations resulting in the modified protein (e.g., missense mutations) may be gradually (when the specific molecular context is achieved) changed to homozygous mutations resulting in the lack of protein during carcinogenesis. Secondly, among different independent pathways of tumorigenesis, those leading to homozygous nonsense mutations are characteristic for cells which are more efficiently stabilized in vitro. To conclude, these observations may be interesting for researchers working with cell line in vitro models illustrating the extent to which they reflect the tumors in vivo.

PIN3 duplication may be partially responsible for TP53 haploinsufficiency.

BACKGROUND: Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon. METHODS: 250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed. RESULTS: Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes. CONCLUSION: TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.

Limited importance of the dominant-negative effect of TP53 missense mutations.

BACKGROUND: Heterozygosity of TP53 missense mutations is related to the phenomenon of the dominant-negative effect (DNE). To estimate the importance of the DNE of TP53 mutations, we analysed the percentage of cancer cases showing a single heterozygous mutation of TP53 and searched for a cell line with a single heterozygous mutation of this gene. This approach was based on the knowledge that genes with evident DNE, such as EGFR and IDH1, represent nearly 100% of single heterozygous mutations in tumour specimens and cell lines. METHODS: Genetic analyses (LOH and sequencing) performed for early and late passages of several cell lines originally described as showing single heterozygous TP53 mutations (H-318, G-16, PF-382, MOLT-13, ST-486 and LS-123). Statistical analysis of IARC TP53 and SANGER databases. Genetic analyses of N-RAS, FBXW7, PTEN and STR markers to test cross-contamination and cell line identity. Cell cloning, fluorescence-activated cell sorting and SSCP performed for the PF-382 cell line. RESULTS: A database study revealed TP53 single heterozygous mutations in 35% of in vivo (surgical and biopsy) samples and only 10% of cultured cells (in vitro), although those numbers appeared to be overestimated. We deem that published in vivo TP53 mutation analyses are not as rigorous as studies in vitro, and we did not find any cell line showing a stable, single heterozygous mutation. G16, PF-382 and MOLT-13 cells harboured single heterozygous mutations temporarily. ST-486, H-318 and LS-123 cell lines were misclassified. Specific mutations, such as R175H, R273H, R273L or R273P, which are reported in the literature to exert a DNE, showed the lowest percentage of single heterozygous mutations in vitro (about 5%). CONCLUSION: We suggest that the currently reported percentage of TP53 single heterozygous mutations in tumour samples and cancer cell lines is overestimated. Thus, the magnitude of the DNE of TP53 mutations is questionable. This scepticism is supported by database investigations showing that retention of the wild-type allele occurs with the same frequency as either nonsense or missense TP53 mutations.

Double hit of NEMO gene in preeclampsia.

The precise etiology of preeclampsia is unknown. Family studies indicate that both genetic and environmental factors influence its development. One of these factors is NFkB, whose activation depends on NEMO (NFkB essential modulator. This is the first study to investigate the association between the existence of single nucleotide variant of the NEMO gene and the appearance of preeclampsia. A total of 151 women (72 preeclamptic women and 79 controls) and their children were examined. Sanger sequencing was performed to identify variants in the NEMO gene in the preeclamptic mothers. The maternal identified variants were then sought in the studied groups of children, and in the maternal and child controls, using RFLP-PCR. Real-time RT-PCR was performed to assess NEMO gene expression in maternal blood, umbilical cord blood and placentas. The sequencing process indicated the existence of two different variants in the 3'UTR region of the NEMO gene of preeclamptic women (IKBKG:c.*368C>A and IKBKG:c.*402C>T). The simultaneous occurrence of the TT genotype in the mother and the TT genotype in the daughter or a T allele in the son increased the risk of preeclampsia development 2.59 fold. Additionally, we found that the configuration of maternal/fetal genotypes (maternal TT/ daughter TT or maternal TT/son T) of IKBKG:c.*402C/T variant is associated with the level of NEMO gene expression. Our results showed that, the simultaneous occurrence of the maternal TT genotype (IKBKG:c.*402C>T variants) and TT genotype in the daughter or T allele in the son correlates with the level of NEMO gene expression and increases the risk of preeclampsia development. Our observations may offer a new insight into the genetic etiology and pathogenesis of preeclampsia.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line.

The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant-EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.

Sensitivity of neoplastic cells to senescence unveiled under standard cell culture conditions.

BACKGROUND: Cancer cells are typically defined as infinitely proliferating, whereas normal cells (except stem cells) are considered as being programmed to become senescent. Our data show that this characterization is misleading. MATERIALS AND METHODS: Multiplex Ligation-dependent Probe Amplification, TP53 sequencing, real-time polymerase chain reaction (PCR) for MUC1 and SCGB2A2 and immunocytochemistry, together with senescence detection assay and real-time microscopic observations were used to analyze primary neoplastic cells isolated from prostate, breast and colorectal tumors, as well as stable cancer cell lines (MCF7, MDA-MB-468, SW962, SK-MEL28, NCI-H1975 and NCI-H469). RESULTS: In all cases of primary cancer cell cultures, in vitro conditions rapidly revealed senescence in the majority of cells. Two out of six stable cancer cell lines did not exhibit any senescence-associated-ß-Galactosidase-positive cells. Interestingly, four cell lines had small sub-populations of senescent cells (single SA-ß-Gal-positive cells). CONCLUSION: Primary neoplastic cells from different types of cancer (prostate, breast, colon cancer) appear to be senescent in vitro. Apparently, cancer cell lines that have been used for many years in drug-testing analyses have constantly been misleading researchers in terms of the general sensitivity of cancer cells to senescence.

Spontaneous in vitro senescence of glioma cells confirmed by an antibody against IDH1R132H.

BACKGROUND: We have recently suggested that glioblastoma cells become spontaneously senescent in cell culture conditions. The antibody specific against IDH1(R132H) offers the perfect opportunity to verify this hypothesis. MATERIALS AND METHODS: We analyzed the features of senescence in 8 glioma cell cultures showing the IDH1(R132H) mutation based on combination of immunocytochemistry, enzymo-cytochemistry, BrdU incorporation assay and real-time microscopic observation. RESULTS: We report that glioma cells showing the IDH1(R132H) mutation become rapidly and spontaneously senescent in vitro. Senescence was observed in both classical and novel serum-free cell culture conditions. Importantly, the senescent IDH1(R132H)-positive cells showed the expression of stemness marker (SOX2). CONCLUSION: In vitro senescence appeared to be the main reason of the difficulties in any kind culturing of glioma cells. 3D cell cultures prolonged the survival and in vitro proliferation of neoplastic IDH1(R132H)-positive cells, however, did not enhance the stabilization efficiency. Senescence of glioma cells is spontaneously triggered in vitro, which offers the opportunity of potential new therapeutic strategies based on this phenomenon.

The failure in the stabilization of glioblastoma-derived cell lines: spontaneous in vitro senescence as the main culprit.

Cell line analysis is an important element of cancer research. Despite the progress in glioblastoma cell culturing, the cells isolated from the majority of specimens cannot be propagated infinitely in vitro. The aim of this study was to identify the processes responsible for the stabilization failure. Therefore, we analyzed 56 primary GB cultures, 7 of which were stabilized. Our results indicate that senescence is primarily responsible for the glioblastoma cell line stabilization failure, while mitotic catastrophe and apoptosis play a minor role. Moreover, a new technical approach allowed for a more profound analysis of the senescent cells in primary cultures, including the distinction between tumor and normal cells. In addition, we observed that glioblastoma cells in primary cultures have a varied potential to undergo spontaneous in vitro senescence, which is often higher than that of the normal cells infiltrating the tumor. Thus, this is the first report of GB cells in primary cell cultures (including both monolayer and spheroid conditions) rapidly and spontaneously becoming senescent. Intriguingly, our data also suggest that nearly half of GB cell lines have a combination of TP53 mutation and CDKN2A homozygous deletion, which are considered as mutually exclusive in glioblastoma. Moreover, recognition of the mechanisms of senescence and mitotic catastrophe in glioblastoma cells may be a step towards a potential new therapeutic approach.

EGFRvIII--a stable target for anti-EGFRvIII therapy.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene alterations play important roles in pathogenesis of glioblastoma. Antibodies against EGFRvIII have been recently developed. Their efficacy depends on numerous factors, including the co-existence of EGFRvIII with other genetic alterations, and especially with point mutations of EGFR. MATERIALS AND METHODS: We examined 91 patients diagnosed with glioblastoma in order to determine the prevalence and mutual relationships between EGFR alterations. Real-time polymerase chain reaction (real-time PCR), fluorescent in situ hybridization (FISH), and sequencing were used to analyze prevalence of the amplification of EGFR gene, polysomy of chromosome 7, EGFRvIII mutation, and point mutations in exons 7-8 and 15 of EGFR. RESULTS: We revealed that all these alterations can occur independently from each other. Nevertheless, the co-existence of EGFRvIII mutation and excessive copies of EGFR gene was observed in most cases (10/14). Similarly, the point mutations in exons 7-8 and 15 co-existed with an excessive number of EGFR copies in nearly all cases. CONCLUSION: EGFRvIII is a reliable and stable target for anti-EGFRvIII therapy.

Screening for EGFR amplifications with a novel method and their significance for the outcome of glioblastoma patients.

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient's age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient's tumour molecular characteristics in the selection of the therapy.