Ken Sasai

Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation

Human cells are known to be more refractory than rodent cells against oncogenic transformation in vitro. To date, the molecular mechanisms underlying such resistance remain largely unknown. The combination of simian virus 40 early region and H-Ras V12 has been effective for transformation of rat embryo fibroblasts, but not for human cells. However, the additional ectopic expression of the telomerase catalytic subunit (hTERT) was reported to be capable of causing transformation of normal human cells. In this study, however, we demonstrate that the combined expression of the above-mentioned three genetic elements is not always sufficient to transform normal human diploid fibroblasts (HDF). Although the expression and function of these introduced genetic elements were essentially the same, among four HDF, TIG-1 and TIG-3 were resistant to transformation. The other two (BJ and IMR-90) showed transformed phenotypes, but they were much restricted compared with rat embryo fibroblasts in expressing simian virus 40 early region and H-Ras V12. In correlation with these phenotypes, TIG-1 and TIG-3 remained diploid after the introduction of these genetic elements, whereas BJ and IMR-90 became highly aneuploid. These results strongly suggest that the lack of telomerase is not the sole reason for the refractory nature of HDF against transformation and that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation.

Promoter hypomethylation regulates CD133 expression in human gliomas.

Brain tumor-initiating cells (BTICs) have been enriched using antibodies against the cell surface protein CD133; however, the biological relevance and the regulatory mechanism of CD133 expression in human gliomas are not yet understood. In this study, we initially demonstrated that CD133 was overexpressed in high-grade human glioblastomas where CD133-positive cells were focally observed as a micro-cluster. In addition, CD133 transcripts with exon 1A, 1B, or 1C were predominantly expressed in glioblastomas. To elucidate the mechanism regulating this aberrant expression of CD133, three proximal promoters (P1, P2, and P3) containing a CpG island were isolated. In U251MG and T98G glioblastoma cells, the P1 region flanking exon 1A exhibited the highest activity among the three promoters, and this activity was significantly inactivated by in vitro methylation. After treatment with the demethylating agent 5-azacytidine and/or the histone deacetylase inhibitor valproic acid, the expression level of CD133 mRNA was significantly restored in glioma cells. Importantly, hypomethylation of CpG sites within the P1, P2, and P3 regions was observed by bisulfite sequencing in human glioblastoma tissues with abundant CD133 mRNA. Taken together, our results indicate that DNA hypomethylation is an important determinant of CD133 expression in glioblastomas, and this epigenetic event may be associated with the development of BTICs expressing CD133.

A Secreted Type of β1,6-N-Acetylglucosaminyltransferase V (GnT-V) Induces Tumor Angiogenesis without Mediation of Glycosylation A NOVEL FUNCTION OF GnT-V DISTINCT FROM THE ORIGINAL GLYCOSYLTRANSFERASE ACTIVITY

Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that β1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.

Careful exclusion of non-neoplastic brain components is required for an appropriate evaluation of O6-methylguanine-DNA methyltransferase status in glioma: relationship between immunohistochemistry and methylation analysis.

Evaluation of O6-methylguanine-DNA methyltransferase (MGMT) expression is important for antiglioma therapy as many clinical trials have demonstrated that promoter hypermethylation and low level expression of MGMT are associated with an enhanced response to alkylating agents. However, here we report that the current strategies used to evaluate MGMT status in gliomas are unreliable. We observed discordance in the MGMT expression status when immunohistochemical evaluation and polymerase chain reaction-based methylation assessments were used: 73% of gliomas with methylated MGMT promoter had substantial numbers of MGMT-immunopositive tumor cells. Furthermore, when MGMT expression was tested in tumor homogenates using reverse transcription-polymerase chain reaction, 43% of tumors were found positive, in comparison to only 24%, when histologic samples were assayed immunohistochemically. To explain these inconsistencies we undertook a detailed immunohistochemical evaluation of tumor samples and found that some gliomas demonstrated remarkably high expression of MGMT in the entire tumor whereas others contained only a small immunopositive area. Additionally, we found that gliomas contained various types of non-neoplastic cells expressing MGMT, including lymphocytes, vascular endothelial cells, and macrophages/microglias, which contribute to overall MGMT expression detected in tumor homogenates, and thus result in overestimation of tumor MGMT expression. Therefore, to correctly establish MGMT expression in the tumor, which could be informative of glioma sensitivity to alkylating agents, exclusion of non-neoplastic brain components from analysis is required.

Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks

BackgroundAn increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene.ResultsA reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression.ConclusionsIn conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.

The Critical Role of the Stem Region as a Functional Domain Responsible for the Oligomerization and Golgi Localization of N-Acetylglucosaminyltransferase V THE INVOLVEMENT OF A DOMAIN HOMOPHILIC INTERACTION

We demonstrated that a region in the stem ofN-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the α-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.

O6-methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies

BackgroundA novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways.ResultsWe established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents.ConclusionNormal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.

The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step

Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N‐acetylglucosaminyltransferase (GnT)‐V transfers a N‐acetylglucosamine residue via a β1,6‐linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT‐V with respect to the bisected oligosaccharide. Although the K m values for the bisected acceptors were comparable to that for a non‐bisected acceptor, the V max values for the bisected acceptors were much lower than that for the non‐bisected acceptor. These findings suggest that the acceptor specificity of GnT‐V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2‐N‐acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.

A population of BJ fibroblasts escaped from Ras-induced senescence susceptible to transformation

Oncogenic stimuli such as H-Ras induce oncogene-induced senescence (OIS) in fibroblasts to protect against transformation. Here we found that a population of the human diploid fibroblasts can escape from OIS induced by H-RasV12. We designated these OIS-escaped cells as OISEC (OIS-escaped cells). OISEC lost the expression of p16 which plays an important role for cell cycle arrest for induction of senescence, but OISEC preserved the p16 expression machinery and exhibited senescence by the treatment with hydrogen peroxide (H(2)O(2)) as stress-induced premature senescence (SIPS). OISEC did not possess anchorage-independent growth potential, and functional disruption of p53 and Rb by SV40 early region encoding large T and small t antigens, induced the aneuploidy phenotype and colony-forming potential of OISEC together with the exhibition of in vivo tumor formation. Finally, we also found that the distinctive feature of OISEC is expression of transcription factors, Oct3/4, SOX2, and Nanog which is closely related to stem-like cell features. This study highlights the presence of a cell population which escaped from OIS, and this OISEC may transform into malignant cancer cells by the additional hits of several genes in vivo.

Clinicopathologic application of lectin histochemistry: bisecting GlcNAc in glioblastoma.

Glycosylation is one of the most common posttranslational modifications and changes in oligosaccharide structures are associated with many human diseases including a number of cancers. Thus, discovering aberrant glycosylation patterns that serve as markers for brain tumor progression and metastasis represents an attractive strategy to improve clinicopathologic diagnosis and to provide aids to the development of novel therapies. To identify glioblastoma (GBM) cells expressing glycoproteins that contain high levels of the bisecting N-acetylglucosamine (GlcNAc) structures, lectin histochemistry was carried out using erythroagglutinating phytohemagglutinin. Although GBM frequently expressed the bisecting GlcNAc, the lectin reactivity varied among tumor regions within individual specimens. Since detailed histopathologic analysis revealed that oligosaccharides with bisecting GlcNAc structures were preferably expressed in tumor regions with low KI67 immunopositivity, immunodetection of the bisecting GlcNAc could be useful to indicate less proliferative regions in human GBM. Our study highlights the potential use of lectin histochemistry to develop new methods for diagnosis that would improve future antiglioma therapy.