Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma.

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene.

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.

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line.

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence.

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods-direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc-in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-ß-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.

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.

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.

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.

Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies.

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor-EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor-EGFRvIII.

Imperfect oligodendrocytic and neuronal differentiation of glioblastoma cells.

Previously, we have reported that glioblastoma (GBM) cells can be differentiated into cells showing neuronal, glial and non-neural (mesenchymal) phenotypes. Before the differentiation the GBM cells co-expressed GFAP, CD44, Beta III tubulin, MAP2, Vimentin, Nestin and SOX-2, whereas during the exposure to a neural differentiation medium the differentiation process was arrested at the early stages and the GBM cells presented features of four phenotypes: multi-lineage, non-neural (mesenchymal), intermediate of neuronal cells and glial cells. Currently, we decided to check if changes in expression of: TH (tyrosine hydroxylase, marker of catecholaminergic cells) and GABA (neurotransmitter of GABAergic neurons) and markers of oligodendrocytic cells (O4, CNP) occur during the exposure of GBM cells to the differentiation medium. After exposure to the PDGF alpha and thyroid hormones (oligodendrocytic differentiation medium 10-30 days) features of oligodendrocytic differentiation were presented by 0.2-2.4% of analyzed cells. During the prolonged neural differentiation (GDNF, bFGF 20-30 days) only few cells showed expression of GABA. Moreover, in our cell cultures, there were not cells expressing markers of catecholaminergic neurons - TH. Our work confirmed that the neuronal differentiation of GBM was inhibited at the stage of the neuronal intermediate phenotype. Moreover, we showed that the oligodendrocytic differentiation of GBM cells is very inefficient.

Earlier onset of Alzheimer's disease: risk polymorphisms within PRNP, PRND, CYP46, and APOE genes.

We studied eight polymorphisms within APOE, PRNP, PRND, and CYP46 genes in 213 Polish late-onset patients with Alzheimer's disease (AD) and 171 non-demented elderly controls. A latent classification approach, grade-of-membership analysis, was taken to identify three extreme pure type risk sets defined by the probabilities of being affected with AD and for genotypes found at the examined genes. Sets I and II represented high intrinsic risk, having a higher density of various genotypes compared to set III, at low intrinsic risk. A gradient of onset age depending on membership in the risk sets was also observed. Logistic regression analysis showed that the highest risk for AD was found for individuals who co-inherited APOE epsilon4 allele, PRNP codon 129 homozygosity, PRND codon 174 Thr allele, and CYP46 rs754203 g allele. AD can be influenced by genetic profiles leading to appearance of the disease, composed of genes which separately evoke a little or unnoticeable effect. Moreover, there may be multiple sufficient risk sets for AD. Looking at multiple genes together rather than analyzing them individually, may improve identification of risk alleles.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors.

BACKGROUND: Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. METHODS: Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. RESULTS: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. CONCLUSION: Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Analysis of APBB2 gene polymorphisms in sporadic Alzheimer's disease.

The accumulation of beta-amyloid (Abeta) in the brain plays a central role in the pathogenesis of Alzheimer's disease (AD). The processing of Abeta precursor protein to Abeta is modulated by binding proteins including APBB2 [amyloid beta precursor protein-binding family B member 2, FE65-like, FE65L1]. We investigated two intronic SNPs within the APBB2 gene: rs13133980 and hCV1558625 (rs17443013), among Polish AD patients and healthy controls (n=213, 171). The frequencies of rs13133980 alleles and genotypes did not differ between cases and controls, irrespective of age of onset or APOE epsilon4 carrier status. The hCV1558625 G allele was over-represented in patients with onset under age 70 compared to controls in the same age range (57% vs. 43%, p=0.03). The association between the hCV1558625 G allele and susceptibility for AD at relatively young ages needs to be confirmed in other samples.

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells.

BACKGROUND: Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells. RESULTS: Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. CONCLUSION: We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

Neuronal and astrocytic cells, obtained after differentiation of human neural GFAP-positive progenitors, present heterogeneous expression of PrPc.

PrP(c) is a cellular isoform of the prion protein with an unknown normal function. One of the putative physiological roles of this protein is its involvement in cell differentiation. Recently, in vitro and in vivo studies showed that GFAP-positive cells have characteristics of stem/progenitor cells that generate neurons and glia. We used an in vitro model of human neurogenesis from GFAP-positive progenitor cells to study the expression of PrP(c) during neural differentiation. Semi-quantitative multiplex-PCR assay and Western blot analysis revealed a significant increase of PRNP expression level in differentiated cells compared to undifferentiated cell population. As determined by immunocytochemistry followed by a quantitative image analysis, the PrP(c) level increased significantly in neuronal cells and did not increase significantly in glial cells. Of note, glial and neuronal cells showed a very large heterogeneity of PrP(c) expression. Our results provide the basis for studying the role of PrP(c) in cell differentiation and neurogenesis from human GFAP-positive progenitor cells.

Regulation of PrPC expression: nerve growth factor (NGF) activates the prion gene promoter through the MEK1 pathway in PC12 cells.

A high expression of PrP(C) in cells is one factor that increases the risk of conversion to the misfolded, disease-associated form (PrP(Sc)) characteristic of transmissible spongiform encephalopathies. Thus, developing a method to control the level of PrP(C) expression in cells could be one way to delay or prevent the onset of clinical signs of these diseases. In this study the mechanisms controlling the expression of the Prnp gene in PC12 cells and in rat brain were examined. We observed a slight activation of a cloned fragment of the human PRNP gene promoter using the luciferase reporter system in PC12 cells stimulated with nerve growth factor (NGF). The activating effect of NGF was enhanced by mitogen-activated protein kinase (MEK1) and suppressed by myristylated serine/threonine kinase (myrAKT). These results suggest that MEK1 is a positive activator of the PRNP promoter that inhibits the AKT pathway. Independent experiments suggested that high expression of PrP(C) in the brain depends on the rate of translation and/or the efficiency of PrP(C) stabilization. We also investigated the epigenic status of the Prnp promoter. We observed no increase of PrP(C) or Prnp mRNA levels in PC12 cells after treatment with the DNA-demethylating agent. The Prnp promoter did not display methylation either in NGF-treated and untreated PC12 cells, or in the rat brain. These results improve the understanding of the regulation of the Prnp gene promoter, a DNA regulatory element controlling the expression of PrP(C), a protein involved in several neurological diseases.

[Analyses of 14-3-3 protein in the cerebrospinal fluid in Creutzfeldt-Jakob disease. Preliminary report]. / Badania bialka 14-3-3 w plynie mózgowo-rdzeniowym w chorobie Creutzfeldta-Jakoba. Doniesienie wstepne.

BACKGROUND AND PURPOSE: Definitive diagnosis of Creutzfeldt-Jakob disease (CJD) is possible only after neuropathological confirmation; there are no non-invasive tests which allow to definitely diagnose CJD pre mortem. The only one biochemical marker included in the diagnostic criteria for CJD approved by WHO is an elevated level of 14-3-3 protein in the cerebrospinal fluid (CSF). The aim of the study was to introduce 14-3-3 analysis for a routine diagnostics of CJD in Poland and to optimise the Western blotting technique used in the Reference Centre of Prion Diseases in Lodz. MATERIAL AND METHODS: We analysed 33 samples of CSF: 17 from patients suspected of CJD (2 definitive, 2 probable, 4 possible, 11 others), and 16 controls (from patients with other neurological diseases). CSF samples were analysed using the Western blotting method and commercially available antibodies (Santa Cruz). We used 2 methods of CSF preparation: with and without lyophilization, and 2 different anti-14-3-3 antibodies: rabbit polyclonal and mouse monoclonal antibodies. RESULTS: We found 14-3-3 protein in both definitive sCJD cases and in one probable case. Among other samples, we obtained a positive result only in one patient with alcohol abuse history and in samples of CSF contaminated with erythrocytes. CONCLUSIONS: The Western blotting test for 14-3-3 protein in CSF is useful for diagnosis of CJD, but only when considered in an appropriate clinical context, together with other diagnostic criteria. Contamination of CSF with erythrocytes can cause false positive results. Analysis of a non-lyophilised CSF and detection of 14-3-3 protein using the mouse monoclonal antibody gave the most reliable results.

CYP46: a risk factor for Alzheimer's disease or a coincidence?

Excess cholesterol is removed from the brain via hydroxylation mediated by cholesterol 24S-hydroxylase (CYP46). Although serum and cerebrospinal fluid (CSF) concentrations of 24S-hydroxycholesterol are altered during the progress of Alzheimer's disease, studies carried out to date in different populations on the association of CYP46 gene polymorphisms and risk of AD have been inconclusive. In this report, we analyzed CYP46 polymorphisms in 215 Polish AD cases and 173 healthy individuals. A fragment of CYP46 intron 2 was amplified by PCR reaction and sequenced. We discovered a new single nucleotide substitution in CYP46 intron 2, but found no difference in particular genotype or allele frequencies between AD patients and controls. However, the GG genotype of the known rs754203 polymorphic site might be a risk factor for AD, especially in APOE varepsilon4 carriers. Interestingly, in AD patients the rs754203 G allele was more frequent in males than in females. However, considering the extreme divergence of results obtained by different authors, a clear connection between the CYP46 gene and AD is questionable.