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Public databases of the Cancer Genome Anatomy Project were used to quantify the relative gene expression levels in glioblastoma multiforme (GBM) and normal brain by Serial Analysis of Gene Expression (SAGE). Analysis revealed HC gp-39 among the genes with the most pronounced changes of expression in tumor cells. Northern hybridization confirmed the results of computer analysis and showed that enhanced expression of the HC gp-39 gene was mainly in GBMs and occasionally in anaplastic astrocytomas. Neither SAGE nor Northern analysis revealed the presence of HC gp-39 mRNA in the glioblastoma cell line, thus the detection of increased quantities of this mRNA in GBMs may be associated with activated macrophages. Since the numbers of infiltrating macrophages and small vessel density are higher in glioblastomas than in anaplastic astrocytomas or astrocytomas, the HC gp-39 gene can be used as a molecular marker in the analysis of malignant progression of astrocytic gliomas. q

BackgroundTemozolomide (TMZ) is a first-line drug for the treatment of glioblastoma. Long-term TMZ-treated tumour cells acquire TMZ resistance by profound reprogramming of the transcriptome, proteome, kinome, metabolism, and demonstrate versatile and opposite changes in proliferation, invasion, in vivo growth, and drug cross-resistance. We hypothesized that chromosomal instability (CIN) may be implicated in the generation of TMZ-driven molecular and phenotype diversity. CIN refers to the rate (cell-to-cell variability) with which whole chromosomes or portions of chromosomes are gained or lost.MethodsThe long-term TMZ-treated cell lines were established in vitro (U251TMZ1, U251TMZ2, T98GTMZ and C6TMZ) and in vivo (C6R2TMZ). A glioma model was achieved by the intracerebral stereotactic implantation of C6 cells into the striatum region of rats. Genomic and phenotypic changes were analyzed by conventional cytogenetics, array CGH, trypan blue exclusion assay, soft agar colony formation assay, scratch wound healing assay, transwell invasion assay, quantitative polymerase chain reaction, and Western blotting.ResultsLong-term TMZ treatment increased CIN-mediated genomic diversity in U251TMZ1, U251TMZ2 and T98GTMZ cells but reduced it in C6TMZ and C6R2TMZ cells. U251TMZ1 and U251TMZ2 cell lines, established in parallel with a similar treatment procedure with the only difference in the duration of treatment, underwent individual phenotypic changes. U251TMZ1 had a reduced proliferation and invasion but increased migration, whereas U251TMZ2 had an enhanced proliferation and invasion but no changes in migration. U251TMZ1 and U251TMZ2 cells demonstrated individual patterns in expression/activation of signal transduction proteins (e.g., MDM2, p53, ERK, AKT, and ASK). C6TMZ and C6R2TMZ cells had lower proliferation, colony formation efficiency and migration, whereas T98GTMZ cells had increased colony formation efficiency without any changes in proliferation, migration, and invasion. TMZ-treated lines demonstrated a differential response to a reduction in glucose concentration and an increased resistance to TMZ re-challenge but not temsirolimus (mTOR inhibitor) or U0126 (MEK1/2 inhibitor) treatment.ConclusionLong-term TMZ treatment selected resistant genotype-phenotype variants or generated novel versatile phenotypes by increasing CIN. An increase of resistance to TMZ re-challenge seems to be the only predictable trait intrinsic to all long-term TMZ-treated tumour cells. Changes in genomic diversity may be responsible for heterogeneous phenotypes of TMZ-treated cell lines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0311-8) contains supplementary material, which is available to authorized users.

Background and Objectives: Our objective was to identify differentially expressed genes involved in the pathogenesis of glioblastoma multiforme (GBM). Methods: Screening of arrayed human fetal brain and human postnatal brain cDNA libraries was performed by differential hybridization with glioblastoma multiforme and human normal brain cDNAs. Results: Repeated differential hybridization of more than 100 cDNA clones selected by primary screening and analysis of RNA from adult normal brain and glial tumors showed 16 nucleotide sequences differentially expressed between normal brain and brain tumors. Among others, decreased content in astrocytic tumors was determined for TSC-22 mRNA corresponding to cDNA in the ICRFp507J1041 clone from human fetal brain cDNA library. Northern blot hybridization of RNA from different human brain tumors showed very low amounts of TSC-22 mRNA in most investigated samples of GBM, anaplastic astrocytoma, and some other tumors. Complete lack of expression of TSC-22 occurred in one sample of anaplastic astrocytoma, as well as in meningioma, brain sarcoma, sarcomatous meningioma, and oligodendroglioma. The differential expression of TSC-22 gene was confirmed by semiquantitative RT-PCR in 15 samples of astrocytomas WHO grade II-IV and three samples of normal brain. Conclusions: Significantly decreased levels of TSC-22 mRNA in human brain and salivary gland tumors and antiproliferative role of TSC-22 strongly suggest a tumor suppressor role for TSC-22.

Screening of human fetal brain cDNA library by glioblastoma (GB) and normal human brain total cDNA probes revealed 80 differentially hybridized clones. Hybridization of the DNA from selected clones and the same cDNA probes confirmed this difference for 38 clones, of which eight clones contained Alu-repeat inserts with increased levels in GB. Thirty clones contained cDNAs corresponding to mitochondrial genes for ATP synthase subunit 6 (ATP6), cytochrome c oxidase subunit II (COXII), cytochrome c oxidase subunit III (COXIII), NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 4 (ND4), and mitochondrial 12S rRNA. The levels of all these mitochondrial transcripts were decreased in glioblastomas as compared to tumor-adjacent histologically normal brain. Earlier we found the same for cytochrome c oxidase subunit I (COXI) Serial Analysis of Gene Expression (SAGE) showed lower content of the tags for all mitochondrial genes in GB SAGE libraries and together with our experimental data could serve as evidence of general inactivation of the mitochondrial genome in glioblastoma-the most malignant and abundant form of human brain tumor. q 2004 Published by Elsevier Ireland Ltd.

More than forty genes with considerably increased expression in glioblastoma as compared to normal human brain were identified by SAGE. One of the most prominent among them was CHI3L2 (YKL-39) gene, which encodes 39 kDa chitinase-like protein.Northern blot hybridization confirmed the data of SAGE for the majority of glioblastomas. Anaplastic astrocytomas could be divided on two groups: in one of them the YKL-39 expression was completely undetectable, but in the other group quite high contents of YKL-39 mRNA were detected. In this study, preliminary data show that patients with undetectable expression of YKL-39 in anaplastic astrocytomas did not have recurrent tumors quite long (more than 2-3 years) period of time. YKL-39 RNA has not been detected in diffuse astrocytomas and in all (but one) samples of normal brain. Increased expression of YKL-39 gene in glioblastomas was shown also at the protein level. Western blots did not shown simultaneous production of YKL-39 and YKL-40, in spite of having high degree of their sequence identity. Increased expression of YKL-39 in subsets of patients with glial tumors, reported here for the first time, together with abnormal increase of the YKL-40 gene expression may be a novel molecular marker for glial tumors.

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