Toshio Ohtsubo

Induction of radioresistance by a nitric oxide-mediated bystander effect.

Abstract Matsumoto, H., Hayashi, S., Hatashita, M., Ohnishi, K., Shioura, H., Ohtsubo, T., Kitai, R., Ohnishi, T. and Kano, E. Induction of Radioresistance by a Nitric Oxide-Mediated Bystander Effect. To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene. Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells. Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium. Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium. Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.

Acidic environment causes apoptosis by increasing caspase activity.

SummaryAn exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2–6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth.

Long-term follow-up results of electrogustometry and subjective taste disorder after middle ear surgery

Objectives The present study compares the long‐term follow‐up results of electrogustometry with patient reports of taste dysfunction after middle ear surgery.

Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells.

Tunicamycin (TM), a naturally occurring antibiotic, blocks the first step in the biosynthesis of N‐linked oligosaccharides in cells. In this study, we investigated whether changes in N‐linked glycosylation affect the sensitivity of head‐and‐neck carcinoma cell lines to cis‐diaminedichloroplatinum(II) (cisplatin) in vitro and in vivo. In vitro treatment of the IMC‐3 and KB cell lines with TM significantly decreased the 50% inhibitory concentration (IC50) of cisplatin, as determined by the MTT assay (24.15 to 10.97 μg/ml, p < 0.05). In addition, TM significantly decreased the IC50 of cisplatin against established cisplatin‐resistant IMC‐3/CR cells (>100 to 14.4 μg/ml, p < 0.05) to levels similar to those against parental IMC‐3 cells. TM treatment decreased the number of Con A‐ and L‐PHA‐binding sites on the surface of tumor cells but had no effect on the intracellular platinum concentration. Induction of apoptosis in vitro by TM plus cisplatin in combination was increased compared with that by cisplatin alone. Furthermore, in vivo administration of TM plus cisplatin in combination significantly inhibited local tumor growth in the cisplatin‐resistant in vivo C3H/He mouse model as compared with the control group (p < 0.05) and increased in vivo apoptosis of tumor cells. Our results suggest that the manipulation of glycosylation by TM in tumor cells might be a useful therapeutic strategy for successful chemotherapy using cisplatin against head‐and‐neck cancer. Int. J. Cancer80:279–284, 1999.

Disruption of mdr1a p-glycoprotein gene results in dysfunction of blood-inner ear barrier in mice.

P-glycoprotein (p-gp), a drug transporter in multidrug-resistant cancer cells, is a transmembrane protein encoded by mdr1a, mdr1b and mdr2 genes in mice. In our previous report, high level p-gp was immunohistochemically detected in capillary endothelial cells of the guinea pig inner ear, supporting a possible role as an extrusion pump in the blood-inner ear barrier (BIB). We investigated the functional involvement of p-gp in the inner ear using mdr1a gene knock-out mice [mdr1a(-/-) mice]. Pharmacokinetic analyses showed that mdr1a(-/-) mice displayed obviously increased accumulations of the p-gp-transported drugs doxorubicin (adriamycin, ADM) and vinblastine in the inner ear tissues compared with those in mdr1a(+/+) mice. Subsequent functional studies using auditory-evoked brainstem responses showed hearing impairment only in mdr1a(-/-) mice after administering these drugs. Furthermore, inhibition of p-gp function by co-administration of cyclosporin A (CsA) with doxorubicin (ADM) in mdr1a(+/+) mice resulted in increased accumulation of ADM in inner ear tissues and hearing impairment similar to that noted in mdr1a(-/-) mice. We conclude that mdr1a p-gp, which acts as an efflux pump in the inner ear, prevents ototoxicity induced by p-gp substrate drugs and contributes to a new functional mechanism in the BIB.

Granulocyte‐colony‐stimulating factor enhances invasive potential of human head‐and‐neck‐carcinoma cell lines

Granulocyte-colony-stimulating factor (G-CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G-CSF also affects nonhematopoietic tumor cells by the binding of G-CSF to its specific receptor (G-CSFR) on the cells. In this study, we investigated the effect of G-CSF on the invasive potential of head-and-neck carcinoma cells, and explored the intracellular events initiated by the binding of G-CSF in tumor cells. In vitro treatment of head-and-neck-carcinoma cell lines, IMC-2, IMC-3, KB, Ca9-22, SCCKN and SCCTF, with recombinant G-CSF (rG-CSF) significantly augmented their invasive potential in dose- and time-dependent manners. Among these cancer cells, IMC-2, IMC-3, KB and Ca9-22 cells produced little G-CSF, while large amounts of G-CSF were produced by SCCKN and SCCTF cell lines. Anti-G-CSF antibody (Ab) abrogated the rG-CSF-enhanced invasiveness to the control level of that in untreated cancer cell lines. Immunocytochemical staining and Western blotting using anti-G-CSFR monoclonal antibody (MAb) revealed the expression of G-CSFR on head-and-neck-cancer cell lines exhibiting the enhancement of invasive activity by rG-CSF. IMC-2 cells, having the highest invasive ability among the cell lines used, showed augmentation of G-CSFR expression on stimulation with rG-CSF. Furthermore, stimulation of IMC-2 cells with rG-CSF induced rapid activation of tyrosine-phosphorylated JAK1, suggesting that the G-CSF signal may be transduced into the cells through G-CSFR. Moreover, the gelatinolytic activity of IMC-2 cells was enhanced by stimulation of rG-CSF, and the enhanced invasiveness was inhibited on addition of the tissue inhibitors of metalloproteinases (TIMPs). These results suggest that exogenous rG-CSF may increase the risk of metastasis and/or local recurrence in patients with G-CSFR-positive head-and-neck squamous-cell carcinoma, via an invasive mechanism.

The effect of sodium thiosulfate on ototoxicity and pharmacokinetics after cisplatin treatment in guinea pigs

The effect of sodium thiosulfate (STS) on the pharmacokinetics and ototoxicity of cisplatin (CDDP) was investigated in guinea pigs. Animals received three intramuscular injections of 7.5 mg/kg CDDP separated by intervals of 5 days with or without STS (1,000 mg/kg) administered intraperitoneally immediately and 1 h after each injection of CDDP or 3 and 6 h later. When administered alone, CDDP caused total outer hair cell (OHC) loss in the basal and second turns of the cochlea. In the group administered CDDP and STS, damage to the OHCs was mild when STS was given concurrently, but was severe when STS was given 3 and 6 h later. Pharmacokinetics measured as free and total platinum (Pt) concentrations in plasma and total Pt concentration in perilymph was not affected after administration of STS with CDDP. These results suggest that an inactive Pt-thiosulfate complex is formed in plasma and is measured as a free Pt component which enters the perilymph via the blood-cochlear barrier. Two possible mechanisms are proposed by which STS reduces ototoxicity: entry of CDDP into target cells such as OHCs and striai marginal cells or binding to intracellular macromolecules of these cells is prevented.

Cell cycle progression and apoptosis after irradiation in an acidic environment

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO·C human colorectal cancer cells expressing wild-type p53 and RC10·1 cells, a subline of RKO·C cells deficient in p53 as well as p53+/+ MEFs and p53−/− MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of γ-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity. Cell Death and Differentiation (2000) 7, 729–738

Morphological and Functional Study of Regenerated Chorda Tympani Nerves in Humans

It is still unclear whether the chorda tympani nerves in humans regenerate after being severed during middle ear surgery, although functional studies have demonstrated recovery of taste 1 to 2 years after surgery. To date, 12 cases of regenerated chorda tympani nerves have been found in our series of patients during secondary surgery. The regenerated nerves of 3 cases of the 12 were removed as samples during secondary surgery to detect regenerated myelinated axons. All regenerated nerves were in the submucosal connective tissue layer of the reconstructed eardrum. In the regenerated nerves, myelinated nerve fibers existed in a small fascicle or in connective tissue, but the number of myelinated axons was low compared with that in normal subjects (1,752 ± 78; n = 3), and the distribution was sparse. The total number of regenerated myelinated axons varied from 141 (8.3%) to 979 (55.9%). From a functional study using electrogustometry, incomplete recovery of electrogustation was observed in all 3 cases before secondary surgery, suggesting that chorda tympani nerves actually regenerate in the middle ear and do function.

Incidence of regeneration of the chorda tympani nerve after middle ear surgery.

We retrospectively reviewed 52 patients who underwent middle ear surgery during which the chorda tympani nerves were severed and who then underwent secondary surgery 1 to 5 years later. In 22 patients (42.3%), regenerated chorda tympani nerves (entire length of the tympanic segment) were detected in the submucosal layer of the reconstructed eardrum during the secondary surgery. Before the secondary surgery, 16 patients (30.8%) showed threshold recovery on electrogustometry. When 5 regenerated nerves were observed by transmission electron microscopy, myelinated nerve fibers were detected in a small fascicle or connective tissue, but the number of myelinated axons was significantly decreased (7.4% to 84.6%; p = .01) compared with that in normal subjects (1,911 ± 324; n = 4). There was a significant difference in the incidence of regeneration between the group with end-to-end anastomosis (5/5 or 100%) and that with nerve gap defects (17/47 or 36.2%; p < .05); this finding suggests that repair of the sectioned nerve produces a better incidence of regeneration than leaving the nerve unrepaired.