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.

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.

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.

Primary breast and prostate epithelial cancer cells may be efficiently cultured in vitro using simple and easily validatable approach–plates coated with a mixture of extracellular matrix components and tissue-specific primary cell medium.

IntroductionReciprocal chromosomal translocations involving one of the IG loci and a proto-oncogene are hallmarks of many types of human B-cell non-Hodgkin lymphomas (B-NHL). 1 As a consequence of such translocations, several proto-oncogenes are placed under the transcriptional control of the active IG locus, causing constitutive expression of the oncogenes with subsequent B-cell transformation. 2 During B-cell tumor progression, IG chromosomal translocations are thought to be early events in the pathogenesis of these neoplasias and result in critical deregulation of multiple oncogenes such as BCL2 t(14;18)(q32;q21) in follicular lymphomas, MYC t(8;14)(q24;q32) in Burkitt lymphomas, CCND1 t(11;14)(q13;q32) in mantle cell lymphomas, MALT1 t(1;14)(p21;q32) in extranodal MALT lymphomas, or BCL6 t(3;V)(q27;V) in diffuse large cell lymphomas. 3 In addition to these chromosomal aberrations, several observations suggest that B-cell receptor (BCR) signaling supplies important tonic survival signals that might be required for the maintenance of B-cell lymphomas. 4 Considering the allelic exclusion model, chromosomal translocation events should occur at equal frequency on the expressed IGH allele and the nonexpressed IGH allele. Because translocations of proto-oncogenes into the IGH-loci are always found on the nonproductively rearranged IGH loci, with few exceptions 5 ; these findings suggest that survival of B-cell lymphoma clones is incompatible with translocations of protooncogenes into the productively rearranged IGH loci because this type of translocation would induce inability to express immunoglobulin (Ig), 2 and suggests the dependency of Ig expression for B-cell lymphoma survival. Moreover, taking into account that the somatic hypermutation machinery induces 2 types of destructive somatic mutations (nonsense mutations and duplications causing reading-frame shifts); these events would lead to the generation of B-cell lymphoma clones that lack Ig expression under nonselective conditions. 6 Because that treatment of patients with anti-idiotypic antibodies (a selective condition) did not result in the selection of Ig-negative B-cell lymphoma clones, 7 this observation also suggests the dependency of Ig expression for the survival of many B-cell lymphomas. 2 In support of this hypothesis, several molecules integrated in the BCR regulatory network appear to act as oncoproteins, such as SYK, 8 ZAP70, 9 BLK, 10 and MALT1, 11 whereas other BCR regulatory molecules surprisingly promote programmed cell death, such as LYN, 12 IP3R, 13 calpains, 14 SLP65, 15 and BTK. 16 These observations exemplify both positive and negative regulatory roles for BCR in signaling and raise the question of how the BCR signal transduction pathway can generate such distinct outcomes and what is the contribution of BCR signaling in B-cell transformation.B-cell antigen receptors specific for self-molecules are normally produced as a consequence of the random VDJ recombination. B cells that recognize a self-antigen undergo programmed cell death ser...