Jun A. Takahashi

Multiple Repair Pathways Mediate Tolerance to Chemotherapeutic Cross-linking Agents in Vertebrate Cells

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor

We report here that a neutralizing mouse monoclonal antibody against basic FGF inhibited both anchorage‐dependent and anchorage‐independent growth of U‐87MG and T98G human glioblastoma cells and HeLa cells, all of which express both the basic FGF and the FGF remptor genes. In addition, the subcutaneous administration of this antibody significantly suppressed the tumor development or these tumor cells in nude mice. Therefore, basic FGF plays an important role in neoplastic growth of these cells. The neutralization or basic FGF will be effective in controlling the growth of tumors, such as glioblastoma and other cancer cells which bear basic FGF and FGF receptors.

Evaluation of primary brain tumors with FLT-PET: usefulness and limitations.

Purpose of the Report: The purpose of this report was to investigate the potential of positron emission tomography using F-18 fluorodeoxythymidine (FLT-PET) in evaluating primary brain tumors. Materials and Methods: FLT-PET was performed in 25 patients with primary brain tumors. FLT uptake in the lesion was semiquantitatively evaluated by measuring the maximal standardized uptake value (SUVmax) and the tumor-to-normal tissue ratio (TNR). SUVmax and TNR were compared with the histologic grade and the expression of the proliferation marker (Ki-67). Results: FLT uptake in normal brain parenchyma was very low, resulting in the visualization of brain tumors with high contrast. Both SUVmax and TNR significantly correlated with the malignant grade of brain gliomas, in which high SUVmax/TNR was obtained for high-grade gliomas. Patients with primary lymphoma also showed SUVmax/TNR equivalent to glioblastoma. There was a positive correlation between SUVmax/TNR and the Ki-67 index. In contrast, spuriously high SUVmax and TNR were obtained in 3 of 6 patients with suspected recurrent tumors (2 patients with recurrent grade 2 glioma and one patient with postoperative granuloma), all of which showed lesion enhancement on MRI after Gd administration. Conclusions: FLT-PET can be used to evaluate the malignant grade and proliferation activity of primary brain tumors, especially malignant brain tumors. However, the presence of benign lesions showing blood–brain barrier disruption cannot be distinguished from malignant tumors and needs to be carefully evaluated.

An early stage mechanism of the age-associated mitochondrial dysfunction in the brain of SAMP8 mice; an age-associated neurodegeneration animal model

In order to characterize the early stage of mitochondrial dysfunction, we investigated the redox state and oxidative phosphorylation of the brain mitochondria from 2-month-old Senescence-accelerated mouse (SAM)P8 and SAMR1 mice; SAMP8 mice exhibit various signs of age-associated neurodegeneration and rapid mitochondrial dysfunction, although SAMR1 mice do not. The redox state was estimated as the reduction rate of Cu-pyruvaldehyde-bis (N4-methylthiosemicarbazone) (Cu-PTSM), the reduction of which is closely related to the electron leakage from the mitochondrial electron transport system in the brain, using electron spin resonance spectrometry (ESRS). The oxidative phosphorylation was measured polarographically. The SAMP8 mouse brain mitochondria demonstrated higher redox state and a higher activity of mitochondrial respiration with lower respiration control ratio than the mitochondria of SAMR1 mouse brains. This indicates that an inefficient hyperactive state can exist in the mitochondrial electron transport system before the age-associated mitochondrial dysfunction develops.

Expression of Fibroblast Growth Factor Receptor-1 in Human Glioma and Meningioma Tissues

We examined the expression of fibroblast growth factor receptor-1 (FGFR-1), namely FLG, in tissues of 18 human gliomas, 10 human meningiomas, 3 human metastatic brain tumors, and 2 normal human brains by means of immunohistochemistry. All tissues were positively stained for FGFR-1. Primary brain tumors were more abundantly immunoreactive than normal brain tissues (Mann-Whitney U test, P < 0.05). There was significant correlation between the expression level of basic fibroblast growth factor (basic FGF) and that of FGFR-1 in tissues of human glioma (Spearmans test, P < 0.05). The expression level of FGFR-1 of tumor cells increased in correlation with that of endothelial cells in glioma tissues (Spearmans test, P < 0.001). We previously reported that basic FGF is produced in more than 90% of human glioma and meningioma tissues. Together with these data, it is suggested that basic FGF is involved in autonomous cell growth and tumorigenesis of gliomas and meningiomas as an autocrine growth factor in vivo.

Gene expression of fibroblast growth factor receptors in the tissues of human gliomas and meningiomas.

Northern blot analysis showed transcripts of two types of the fibroblast growth factor (FGF) receptor genes, flg and bek, in almost all the tissues samples of 18 human gliomas and 22 human meningiomas, which produced abundant basic and/or acidic FGF. From immunohistochemistry, FGF receptors were expressed in the tumor cells of a glioma and a meningioma. RNA expression of these FGF receptors was also detectable in normal human brains and normal bovine meninges. The expression level of either FGF receptor gene was not significantly different between tumor tissues and normal tissues.

EFFECT OF EARLY OPTIC CANAL UNROOFING ON THE OUTCOME OF VISUAL FUNCTIONS IN SURGERY FOR MENINGIOMAS OF THE TUBERCULUM SELLAE AND PLANUM SPHENOIDALE

OBJECTIVEThe aim of this study was to evaluate the effect of early optic canal unroofing on visual function in patients with meningiomas of the tuberculum sellae and planum sphenoidale. METHODSWe retrospectively reviewed the clinical records of 20 consecutive patients with tuberculum sellae meningiomas and two patients with planum sphenoidale meningiomas who were admitted to our institution from 1999 to 2007. Factors that may influence postoperative visual functions were analyzed, including patients age and sex, duration of preoperative visual symptoms, preoperative visual acuity, tumor size, tumor consistency, tumor extension into the optic canal, tumor adhesion to the optic nerve, timing of optic canal unroofing, and tumor resection rate. RESULTSThe mean patient age was 52.9 ± 13.7 years (range, 27–73 yr); 18 patients were women and four were men. The mean maximum tumor size was 2.3 ± 0.7 cm (range, 1.5–3.5 cm). Visual symptoms were present preoperatively in 19 patients, and three patients were asymptomatic. The mean duration of visual symptoms was 12.0 ± 16.4 months (range, 0–72 mo). Tumor resection was evaluated according to Simpsons grade, and Grade II was achieved in 14, Grade III in two, and Grade IV in six (two patients were recurrent cases). Tumors were extended into the optic canal in 15 patients, and severe adhesion to the optic nerve was observed in nine patients. Tumor consistency was soft in eight patients, intermediate in eight patients, and hard in six patients. The optic canal was unroofed early before dissection or manipulation of tumor in nine patients (early group) and after dissection of tumor in seven patients (late group), and optic canal unroofing was not performed in six patients (none group; no canal extension in two and intentional incomplete resection in four patients). Visual symptoms were improved in 10 patients, unchanged in seven patients, and worsened in five patients (transient in two and permanent in three). Logistic regression analysis revealed that early optic canal unroofing was an independent factor for postoperative improvement of visual symptoms. CONCLUSIONEarly optic canal unroofing may increase the possibility of improved preoperative visual symptoms in surgical resection of tuberculum sellae meningiomas and planum sphenoidale meningiomas.

Apoptosis induced by selenium in human glioma cell lines

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced the typical ladder pattern of DNA fragmentation commonly found in apoptosis), which were prevented by catalase. Few effects of selenium on NTI4 fibroblasts were found. These findings demonstrate that selenium may induce, by apoptosis, cell death of human glioma cell lines, which are resulting from free radical oxygen forming.

Intradural en-bloc removal of the anterior clinoid process

SummaryBackground. Anterior clinoidectomy is useful in the surgical treatment of paraclinoid and parasellar lesions. Previously reported procedures require expertise in drilling, the alternative method reported here reduces the drilling procedure. Methods and results. En-bloc clinoidectomy is performed intradurally via the standard pterional approach. A 1 to 2 mm-wide narrow drill line is placed with a 1 mm-wide diamond burr through the lesser sphenoid wing. It encircles the medial border of the optic canal and the lateral border of the lesser sphenoid wing over the superior orbital fissure, and is located about 1 cm anterior to the posterior margin of the optic canal. After drilling, one bony piece that includes the anterior clinoid process (ACP) and the optic canal roof remains connected to the basisphenoid bone by the optic strut alone. The optic strut is then fractured easily by applying leverage near its junction with the basisphenoid bone and the piece is removed en bloc with the major part of the optic strut, requiring little or no additional drilling of the residual bony fragments.Of 37 patients who underwent our en-bloc clinoidectomy, only one suffered complications consisting of cerebrospinal fluid (CSF) leakage through the sphenoid sinus. Our procedure requires an average of 20 min. Conclusions. Intradural en-bloc removal of the ACP with fracture of the optic strut requires minimal drilling, resulting in decreased risk of injury to the optic nerve and a shortened time for clinoidectomy.

Endogenous tenascin‐C enhances glioblastoma invasion with reactive change of surrounding brain tissue

Tenascin‐C is an extracellular matrix glycoprotein implicated in embryogenesis, wound healing and tumor progression. We previously revealed that tenascin‐C expression is correlated with the prognosis of patients with glioblastoma. However, the exact role of endogenous tenascin‐C in regulation of glioblastoma proliferation and invasion remains to be established. We show here that endogenous tenascin‐C facilitates glioblastoma invasion, followed by reactive change of the surrounding brain tissue. Although shRNA‐mediated knockdown of endogenous tenascin‐C does not affect proliferation of glioblastoma cells, it abolishes cell migration on a two‐dimensional substrate and tumor invasion with brain tissue changes in a xenograft model. The tyrosine phosphorylation of focal adhesion kinase, a cytoplasmic tyrosine kinase that associates with integrins, was decreased in tenascin‐C‐knockdown cells. In the analysis of clinical samples, tenascin‐C expression correlates with the volume of peritumoral reactive change detected by magnetic resonance imaging. Interestingly, glioblastoma cells with high tenascin‐C expression infiltrate brain tissue in an autocrine manner. Our results suggest that endogenous tenascin‐C contributes the invasive nature of glioblastoma and the compositional change of brain tissue, which renders tenascin‐C as a prime candidate for anti‐invasion therapy for glioblastoma. (Cancer Sci 2009)