OCT4 Expression in Gliomas Is Dependent on Cell Metabolism.

The OCT4 transcription factor is necessary to maintain cell stemness in the early stages of embryogenesis and is involved in the formation of induced pluripotent stem cells, but its role in oncogenesis is not yet entirely clear. In this work, OCT4 expression was investigated in malignant gliomas. Twenty glioma cell lines and a sample of normal adult brain tissue were used. OCT4 expression was found in all studied glioma cell lines but was not detected in normal adult brain tissue. For one of these lines, OCT4 knockdown caused tumor cell death. By varying the culture conditions of these cells, we unexpectedly found that OCT4 expression increased when cells were incubated in serum-free medium, and this effect was significantly enhanced in serum-free and L-glutamine-free medium. L-glutamine and the Krebs cycle, which is slowed down in serum-free medium according to our NMR data, are sources of α-KG. Thus, our data indicate that OCT4 expression in gliomas may be regulated by the α-KG-dependent metabolic reprogramming of cells.

Abnormal activity of transcription factors gli in high-grade gliomas.

Malignant transformation is associated with loss of cell differentiation, anaplasia. Transcription factors gli, required for embryonic development, may be involved in this process. We studied the activity of transcription factors gli in high-grade gliomas and their role in maintenance of stem cell state and glioma cell survival. 20 glioma cell lines and a sample of a normal adult brain tissue were used in the present study. We found the expression of gli target genes, including GLI1 and FOXM1, in all tested glioma cell lines, but not in the normal tissue. Interestingly, the expression of gli target genes in some glioma cell lines was observed together with a high level of their transcriptional repressor, Gli3R. Knockdown of GLI3 in one of these lines resulted in decrease of gli target gene expression. These data suggest that Gli3R does not prevent the gli target genes transcription, and gli3 acts in glioma cells more as an activator, than a repressor of transcription. We observed that gli regulated the expression of such genes, as SOX2 or OCT4 that maintain stem cell state, and TET1, involving in DNA demethylation. Treatment with GANT61 or siRNA against GLI1, GLI2, or GLI3 could result in complete glioma cell death, while cyclopamine had a weaker and line-specific effect on glioma cell survival. Thus, the gli transcription factors are abnormally active in high-grade gliomas, regulate expression of genes, maintaining the stem cell state, and contribute to glioma cell survival.

The rare nonsense mutation in p53 triggers alternative splicing to produce a protein capable of inducing apoptosis.

P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126. The mutation eliminates the recognition site for the restriction endonuclease Sca I that allowed us to carry out RFLP analysis of DNA extracted from the clinical samples and suggests that this mutation is more frequent than is documented in the p53 databases. Both the ECV-304 and EJ cell lines, that probably originate from the bladder carcinoma T24 cell line, were confirmed to contain the homozygous 378C>G mutation but were shown to produce the p53 protein of expected full-length size detected by Western blotting. We provide evidence that the 378C>G mutation generates an alternative 3' splice site (ss) which is more often used instead of the authentic upstream 3' ss, driving the production of mRNA encoding the protein with the single amino acid deletion (p53ΔY126). Using endogenous expression, we demonstrated that the p53ΔY126 protein is nearly as active as the wild type protein in inducing the p21/Waf1 expression and apoptosis.

Exosomes are natural carriers of exogenous siRNA to human cells in vitro.

BACKGROUND: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies. RESULTS: This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells. CONCLUSIONS: The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy.