Step-wise and punctuated genome evolution drive phenotype changes of tumor cells.

The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1_CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa_CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293_pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293_CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293_CHI3L1 and HeLa_CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by genome context. Temozolomide treatment of 293_pcDNA3.1 cells intensified the stochastic punctuated genome changes and CNAs, and significantly reduced viability and CFE. In contrast, temozolomide treatment of HeLa_CHI3L1 cells promoted the step-wise genome changes, CNAs, and increased viability and CFE, which did not correlate with the ectopic CHI3L1 production. Thus, consistent coevolution of karyotypes and phenotypes was observed. CIN as a driving force of genome evolution significantly influences growth characteristics of tumor cells and should be always taken into consideration during the different experimental manipulations.

Bradykinin antagonists and thiazolidinone derivatives as new potential anti-cancer compounds.

Glioblastoma (GB), the most aggressive brain tumour, and mantle cell lymphoma (MCL), a rare but very aggressive type of lymphoma, are highly resistant to chemotherapy. GB and MCL chemotherapy gives very modest results, the vast majority of patients experience recurrent disease. To find out the new treatment modality for drug-resistant GB and MCL cells, combining of bradykinin (BK) antagonists with conventional temozolomide (TMZ) treatment, and screening of thiazolidinones derivatives were the main objectives of this work. As it was revealed here, BKM-570 was the lead compound among BK antagonists under investigation (IC50 was 3.3 µM) in human GB cells. It strongly suppressed extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation. BK antagonists did not decrease the viability of MCL cells, thus showing the cell-specific mode, while thiazolidinone derivatives, a novel group of promising anti-tumour compounds inhibited proliferation of MCL cells: IC50 of ID 4526 and ID 4527 compounds were 0.27 µM and 0.16 µM, correspondingly. However, single agents are often not effective in clinic due to activation of collateral pathways in tumour cells. We demonstrated a strong synergistic effect after combinatorial treatment by BKM-570 together with TMZ that drastically increased cytotoxic action of this drug in rat and human glioma cells. Small proportion of cells was still viable after such treatment that could be explained by presence of TMZ-resistant cells in the population. It is possible to expect that the combined therapy aimed simultaneously at different elements of tumourigenesis will be more effective with lower drug concentrations than the first-line drug temozolomide used alone in clinics.

Chitinase 3-like 2 protein monoclonal antibodies.

Chitinase 3-like 2 (CHI3L2) is one of the most overexpressed genes in glioblastoma. Despite this, both the CHI3L2 gene and its protein product CHI3L2 are poorly characterized. Here we report the generation and characterization of monoclonal antibodies to CHI3L2 protein (CHI3L2 MAbs). Bacterially expressed 6 His-tagged full-length CHI3L2 was used as antigen. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells. Hybridoma clones 2D3 and 4D2 producing high titer CHI3L2 MAbs were identified by enzyme-linked immunosorbent assay (ELISA) and further examined for their activity with the CHI3L2 protein by Western blot analysis and immunoprecipitation. The 2D3 clone was chosen for mouse inoculation and ascites formation. Antibodies derived from the ascitic fluid specifically recognized the recombinant CHI3L2 protein and strongly interacted with CHI3L2 in glioblastoma tissue lysate, as determined by Western blot analysis. The antibodies generated may be useful as a tool in various aspects of CHI3L2 investigation.

Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation.

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.

Immortalized cells and one oncogene in malignant transformation: old insights on new explanation.

BACKGROUND: Nearly thirty years ago, it was first shown that malignant transformation with single oncogene necessarily requires the immortal state of the cell. From that time this thesis for the cells of human origin was not disproved. The basic point which we want to focus on by this short communication is the correct interpretation of the results obtained on the widely used human embryonic kidney 293 (HEK293) cells. RESULTS: Intensive literature analysis revealed an increasing number of recent studies discovering new oncogenes with non-overlapping functions. Since the 1970s, dozens of oncogenes have been identified in human cancer. Cultured cell lines are often used as model systems in these experiments. In some investigations the results obtained on such cells are interpreted by the authors as a malignant transformation of normal animal or even normal human cells (as for example with HEK293 cells). However, when a cell line gains the ability to undergo continuous cell division, the cells are not normal any more, they are immortalized cells. Nevertheless, the authors consider these cells as normal human ones, what is basically incorrect. Moreover, it was early demonstrated that the widely used human embryonic kidney 293 (HEK293) cells have a relationship to neurons. CONCLUSIONS: Thus, the experiments with established cell lines reinforce the notion that immortality is an essential requirement for malignant transformation that cooperates with other oncogenic changes to program the neoplastic state and substances under such investigation should be interpreted as factors which do not malignantly transform normal cells alone, but possess the ability to enhance the tumorigenic potential of already immortalized cells.

Gene Encoding Chitinase 3-Like 1 Protein (CHI3L1) is a Putative Oncogene.

An important task in understanding oncogenesis is the identification of those genes whose copy number and expression increase during tumorigenesis. Previously, in an effort to identify genes which could be used as molecular markers for glial tumors, we compared gene expression in glioblastoma to the normal brain cells. Among the genes with the most pronounced increased expression in tumors there was CHI3L1, encoding the secreted chitinase 3-like 1 protein (also known as HC gp-39 or YKL-40). Expression of CHI3L1 was found increased significantly in various tumors in comparison with corresponding normal tissues. Here we show that CHI3L1 can decrease the doubling time of 293 cells. We have also demonstrated that CHI3L1 allows the anchorage-independent growth in soft agar and, in addition, stable CHI3L1 expression made 293 cells tumorigenic: these cells stimulate the initiation of tumors after their xenograft transplantation into the Wistar rat brains. Thus, the overexpression of CHI3L1 is likely to be critical in the development of some tumors and when we gain more information about mechanisms of CHI3L1 oncogenicity, it could be used as one of the potential targets for anticancer therapy.

No association of (-131C-->G) variant of CHI3L1 gene with risk of glioblastoma and prognosis.

Expression of CHI3L1 (YKL-40) has been correlated with prognosis of glioblastoma. The variant allele (-131C-->G) of CHI3L1 promoter results in a lower transcription of CHI3L1. Therefore, we tested the hypothesis that the G variant could protect against the risk of gliomas or have a favorable prognostic impact. DNA from 296 glioblastoma patients and 190 controls were genotyped on the -131 allele. Tumor RNA was obtained from 108 patients for CHI3L1 transcript quantification. Neither genotype nor allele distribution differed between patients and controls. There was no significant difference in survival between the CC, CG, and GG patients despite the few GG patients tended to have a longer survival. There was no correlation between genotype and CHI3L1 expression in tumor samples. Taken together our data suggest that the variant allele (-131C-->G) of CHI3L1 promoter has no significant impact on survival and is not a prognostic factor for glioblastoma.

Reduction of the transcription level of the mitochondrial genome in human glioblastoma.

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.

Peculiarities of molecular events in human glial tumors revealed by serial analysis of gene expression (SAGE).

AIM: To enhance glioblastoma (GB) marker discovery we compared gene expression in glioblastoma with human normal brain (NB). METHODS: Serial analysis of gene expression (SAGE) was used to determine the genes differentially expressed between glioblastoma and human NB by accessing SAGEmap NCBI web site. RESULTS: Of 117 genes with more than 5-fold difference (P less, not similar 0.05) found by comparison of five GB vs. two NB SAGE libraries, 24 had an increased expression in GB. High expression of 10 genes in GB as well as in GB cell lines suggested that expression in the bulk tumors originated from transformed cells. CONCLUSION: Since the highest expression levels of these 24 genes were mostly found in GB, they can be viewed as molecular markers in the analysis of malignant progression of astrocytic tumors.

HC gp-39 gene is upregulated in glioblastomas.

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.

Downregulation of putative tumor suppressor gene TSC-22 in human brain tumors.

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. J.

Complete nucleotide sequence of the chum salmon insulin-like growth factor II gene.

Insulin-like growth factor II (IGF-II) is thought to play an important role in development and growth of vertebrates. In contrast to mammals, the IGF-II gene is expressed at a high level from the early stages of embryonic development until the adult stage and IGF-II peptide is produced in virtually all organs of bony fish, indicating a physiological importance of IGF-II 'under water'. Therefore, we describe here the complete nucleotide sequence (accession no. X97225) and organization of the chum salmon IGF-II gene. In addition, the phylogenetic relationship of the IGF-II hormones is analysed. Although the chum salmon contains two non-allelic insulin and IGF-I genes, only one IGF-II gene could be identified. The chum salmon IGF-II gene consists of four exons and is comprised of 7992 bp from the putative transcription initiation site to the poly(A) site. Activation of the only promoter of the salmon IGF-II gene gives rise to a single 4 kb transcript. The fish IGF-II gene is much smaller and simpler organized than its known mammalian counterparts that are governed by several tissue-specific and developmental stage-dependent promoters. All known mammalian IGF-II genes to date have been found to form a conserved linkage group with the insulin and tyrosine hydroxylase (TH) genes and are organized as TH-insulin-IGF-II genomic locus. However, in our study we could find no linkage between the insulin and IGF-II genes, or between the insulin, TH and IGF-II genes, at least within approximately 20 kb of the chum salmon IGF-II genomic sequence. In spite of minor differences, the overall organization of the IGF-II genes turned out to be very similar in bony fish. A limited analysis of the phylogenetic relationship between IGF-II prohormones indeed showed a very conservative phylogenesis of IGF-II in bony fish that may indicate the particular significance of IGF-II in these animals.