Sylwester Piaskowski

Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.

Background:It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established.Methods and results:IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material.Conclusion:The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer

ObjectiveTo analyse the single-nucleotide polymorphisms (SNPs): ABCB11236C>T, ABCB12677G>T/A, ABCB13435C>T and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.Materials and methodsTumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.ResultsA statistically significant higher frequency of T1236 allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Coxs proportional hazard model indicated that the T1236 allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C3435 allele and significant differences in the C3435 allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T1236-G2677-C3435 only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).ConclusionsThese results suggest association between T1236 allele and T1236-G2677-C3435 haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.

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

BackgroundAlthough 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.MethodsFollowing 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.ResultsIn 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.ConclusionOur 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.

Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1) and risk of multiple myeloma

MDR1(ABCB1) gene encodes for P-glycoprotein (P-gp, MDR1, ABCB1), an ATP-binding cassette superfamily member involved in the transport of xenobiotics. Here, we investigated whether common MDR1 single nucleotide polymorphisms (1236C>T, 2677G>A/T and 3435C>T) affect predisposition to multiple myeloma. Genotyping was performed in 111 myeloma patients and 96 controls by PCR-based assays. Haplotypes were inferred using PHASE algorithm. We found comparable allele and genotype frequencies among myeloma patients and controls. Moreover, patient and control groups did not differ regarding MDR1 haplotype distribution (p=0.18). In conclusion, our results do not support major influence of MDR1 variants on the risk of myeloma in Caucasians.

Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system

IntroductionThe prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. Generation of iPSCs from somatic cells has opened up new possibilities to investigate stem cell biology, to better understand pathophysiology of human diseases, and to design new therapy approaches in the field of regenerative medicine. In this study, we focus on the ability of the episomal system, a non-viral and integration-free reprogramming method to derive iPSCs from somatic cells of various origin.MethodsCells originating from neonatal and adult tissue, renal epithelium, and amniotic fluid were reprogrammed by using origin of replication/Epstein-Barr virus nuclear antigen-1 (oriP/EBNA-1)-based episomal vectors carrying defined factors. The iPSC colony formation was evaluated by using immunocytochemistry and alkaline phosphatase assay and by investigating gene expression profiles. The trilineage formation potential of generated pluripotent cells was assessed by embryoid body-mediated differentiation. The impact of additionally introduced factors on episome-based reprogramming was also investigated.ResultsReprogramming efficiencies were significantly higher for the epithelial cells compared with fibroblasts. The presence of additional factor miR 302/367 in episomal system enhanced reprogramming efficiencies in fibroblasts and epithelial cells, whereas the downregulation of Mbd3 expression increased iPSC colony-forming efficiency in fibroblasts solely.ConclusionsIn this study, we performed a side-by-side comparison of iPSC colony-forming efficiencies in fibroblasts and epithelial cells transiently transfected with episomal plasmids and demonstrated that iPSC generation efficiency was highest when donor samples were derived from epithelial cells. We determined that reprogramming efficiency of episomal system could be further improved. Considering results obtained in the course of this study, we believe that episomal reprogramming provides a simple, reproducible, and efficient tool for generating clinically relevant pluripotent 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.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

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.

Reduced expression of ELAVL4 in male meningioma patients

Meningioma is a frequently occurring tumor of the central nervous system. Among many genetic alternations, the loss of the short arm of chromosome 1 is the second most frequent chromosomal abnormality observed in these tumors. Here, we focused on the previously described and well-established minimal deletion regions of chromosome 1. In accordance with the Knudson suppressor theory, we designed an analysis of putative suppressor genes localized in the described minimal deletion regions. The purpose was to determine the molecular background of the gender-specific occurrence of meningiomas. A total of 149 samples were examined for loss of heterozygosity (LOH). In addition, 57 tumor samples were analyzed using real-time polymerase chain reaction. We examined the association between the expression of selected genes and patient age, gender, tumor grade and presence of 1p loss. Furthermore, we performed an analysis of the most stable internal control for real-time analysis in meningiomas. LOH analysis revealed gender-specific discrepancies in the frequency of 1p aberrations. Moreover, statistical correlation between the gene expression level and gender was significant for the ELAVL4 gene as we found it to be lower in males than in females. We conclude that meningiomas present different features depending on patient gender. We suggest that ELAVL4 can be involved in the pathogenesis of meningiomas in male patients.

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.