Biomedical subjects

Kazunori Yukawa

Publications and source records attributed to Kazunori Yukawa.

At least 19 recordsLinked to original sources

PubMed2006

Impaired postnatal development in C/EBPbeta-deficient mice.

Tail DNA genotyping of fetal and neonatal mice from C/EBPbeta heterozygous parents was performed to determine whether the decreased number of surviving C/EBPbeta mutants was caused by prenatal or postnatal death. Eighty-four 3-week-old mice born of heterozygous parents had significantly lower numbers of C/EBPbeta-deficient offspring than the expected Mendelian ratio (29.8%+/+, 65.5%+/-, 4.76%-/-, P<0.05). The genotypes of 72 fetal mice from intercrossed heterozygotes showed approximately the expected 1:2:1 Mendelian ratio (18.1% +/+, 52.8% +/-, 29.2% -/-, P>0.05). No difference in the proportions by sex could be detected in these perinates. This data indicates that C/EBPbeta-deficient mice have unknown lethal problems between the embryonic stage and weaning.

Animals↗

PubMed2006

Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis.

Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses. The primary receptors for semaphorins are members of the plexin family. In particular, plexin-A1, together with ligand-binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins, whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins. Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system. However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1(-/-)) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem-2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin-A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins.

Adaptor Proteins, Signal Transducing↗

PubMed2006

A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells.

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.

Antineoplastic Agents↗

PubMed2006

Optimal combination chemotherapy and chemoradiotherapy with etoposide for advanced cervical squamous cancer cells in vitro.

Patients with unresectable advanced carcinoma of the uterine cervix are usually treated with chemotherapy or chemoradiotherapy. In the present study, the optimal administration protocol for etoposide in chemotherapy and chemoradiotherapy for advanced cervical cancer patients was investigated in vitro using the radio-sensitive and anticancer drug-sensitive human cervical squamous cell carcinoma cell line ME180. Therapeutic doses of concurrent irradiation reduced the cellular etoposide sensitivity in a dose-dependent manner, while postirradiation-surviving subclones established from repeatedly irradiated ME180 cells showed significantly higher etoposide sensitivities than the non-irradiated parent cells. Of the 6 monoclonal etoposide-resistant subclones established from ME180 cells, 5 were significantly radioresistant. Although the etoposide-resistant subclones were also significantly resistant to other anticancer drugs, such as cisplatin, carboplatin, nedaplatin, pirarubicin, paclitaxel and docetaxel, they were more sensitive to 5-fluorouracil, mitomycin C and SN38 than the parent cells. Flow cytometric analyses revealed that the etoposide-resistant subclones showed significantly increased cell surface expression of CD40 compared to the parent cells, which expressed undetectable levels of CD40. However, the expression of some integrin receptor subunits, such as CD29, CD49a and CD49f, was apparently reduced in the etoposide-resistant subclones. These results indicate that etoposide should be administered to advanced cervical squamous cancer patients after the completion of radiotherapy, rather than as a concurrent chemoradiotherapy. In order to kill surviving etoposide-resistant cancer cells more effectively, 5-fluorouracil, mitomycin C and irinotecan may be candidate combination drugs for use with etoposide. Differential expression of integrin receptors and CD40 may be involved in the acquisition of etoposide resistance by cervical squamous cancer cells.

Antigens, CD20↗

PubMed2006

Deletion of the kinase domain from death-associated protein kinase enhances spatial memory in mice.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. DAPK is highly expressed in hippocampal neurons which are essential elements for memory formation. To examine if DAPK is implicated in spatial learning and memory, both wild-type and DAPK-mutant mice were subjected to Morris water maze tests. DAPK-mutant mice were generated by deleting 74 amino acids from the catalytic kinase domain of DAPK, and were used to investigate roles of the DAPK kinase domain in regulating spatial memory. Both mutant and wild-type mice were able to learn the water maze tasks to locate a hidden escape platform. In the first probe test, mutant mice showed a more precise memory for platform position compared to wild-type mice. In the reversal training in which the platform was located opposite from the original position, DAPK-mutant mice exhibited superior spatial learning compared to wild-type mice. DAPK-mutant mice also showed a more precise memory than their wild-type littermates in the probe trial of reversal test. Thus, the present results revealed crucial implications of DAPK in regulating spatial memory in mice.

Animals↗

PubMed2005

Hypotensive effect of des-acyl ghrelin at nucleus tractus solitarii of rat.

Ghrelin is a gut-brain peptide and its endocrine activities are mediated by GH secretagogue receptor (GHSR)-1a. Des-acyl ghrelin does not activate GHSR-1a and is devoid of endocrine activities. While the microinjection of ghrelin into rat nucleus tractus solitarii (NTS) elicited hypotensive effects, this was not the case upon injection into GHSR-expressing rostral ventrolateral medulla or caudal ventrolateral medulla. To make clear the reason of the discrepancy between receptor distribution and neuronal responses, we examined the cardiovascular response of rats microinjected with des-acyl ghrelin into NTS. Intra-NTS injection of des-acyl ghrelin significantly reduced mean arterial pressure and heart rate. The hypotensive and bradycardic activity evoked by des-acyl ghrelin was not significantly different from that of native ghrelin. These results suggest that des-acyl ghrelin contribute to the regulation of cardiovascular control and that a receptor other than GHSR-1a exists in NTS.

Animals↗

PubMed2005

Direct in vivo protein transduction into a specific restricted brain area in rats.

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.

Animals↗

PubMed2005

Nonredundant roles of Sema4A in the immune system: defective T cell priming and Th1/Th2 regulation in Sema4A-deficient mice.

The class IV semaphorin Sema4A provides a costimulatory signal to T cells. To investigate the possible developmental and regulatory roles of Sema4A in vivo, we generated Sema4A-deficient mice. Although Sema4A-deficient mice develop normally, DCs and T cells from knockout mice display poor allostimulatory activities and T helper cell (Th) differentiation, respectively. Interestingly, in addition to its expression on DCs, Sema4A is upregulated on Th1-differentiating cells, and it is necessary for in vitro Th1 differentiation and T-bet expression. Consequently, in vivo antigen-specific T cell priming and antibody responses against T cell-dependent antigens are impaired in the mutant mice. Additionally, Sema4A-deficient mice exhibit defective Th1 responses. Furthermore, reconstitution studies with antigen-pulsed DCs reveal that DC-derived Sema4A is important for T cell priming, while T cell-derived Sema4A is involved in developing Th1 responses. Collectively, these results indicate a nonredundant role of Sema4A not only in T cell priming, but also in the regulation of Th1/Th2 responses.

Animals↗

PubMed2005

The kinase domain of death-associated protein kinase is inhibitory for tubulointerstitial fibrosis in chronic obstructive nephropathy.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We have shown that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. DAPK-mutant mice, generated by deletion of 74 amino acids from the catalytic kinase domain, were used to investigate the role of the DAPK kinase domain in renal fibrosis following COU. Interstitial collagen and alpha-smooth muscle actin (alpha-SMA) expressions in situ were compared between obstructed kidneys in wild-type and mutant mice. As a result, tubulointerstitial fibrosis, as quantified by interstitial collagen expression, was significantly augmented in mutant kidneys compared with wild-type kidneys following COU. Furthermore, deletion of the kinase domain from DAPK significantly increased the appearance of alpha-SMA-positive myofibroblasts in the renal interstitium during COU. Thus, our results suggest that the kinase domain deleted by gene targeting plays a suppressive role for the development of renal fibrosis through inhibition of the tubular epithelial-to-mesenchymal transition in a mouse model of COU.

Actins↗

PubMed2005

STAT6 deficiency inhibits tubulointerstitial fibrosis in obstructive nephropathy.

To elucidate the contribution of signal transducer and activator of transcription (STAT) 6 to the pathophysiology of chronic renal injury, STAT6-/- mice were subjected to unilateral ureteral ligation together with wild-type control mice. STAT6-/- kidneys had more apoptotic cells and a greater influx of F4/80-positive cells than wild-type kidneys following ureteral obstruction. There was a much larger alpha-smooth muscle actin-positive area in STAT6-/- kidneys than in wild-type kidneys after ureteral ligation. However, renal fibrosis, as quantified by Masson-Trichrome staining, was not significantly exaggerated in STAT6-/- kidneys compared with wild-type kidneys. The accumulation of collagen I was significantly less in STAT6-/- kidneys than in wild-type kidneys. These observations indicate that the STAT6 signal transduction pathway exerts a protective role on renal cell apoptosis in chronic obstructive uropathy. Our findings also suggest that the STAT6 pathway may have a promotive effect on renal fibrosis by activating collagen synthesis following ureteral obstruction.

Actins↗

PubMed2005

Down-regulation of dopamine transporter and abnormal behavior in STAT6-deficient mice.

To determine the role of STAT6 transcription factors in brain function, we performed a battery of mouse behavioral analyses of STAT6-deficient mice and made comparisons with wild-type mice. STAT6-deficient mice were significantly more hyperactive in the final two blocks of an open field test, indicating abnormal habituation in completing the task. Two learning tasks, a water maze and a passive avoidance test, were mastered by STAT6-deficient mice as effectively as the wild-type. RT-PCR analysis suggested that the levels of dopamine transporter (DAT) mRNA may be lower in the midbrain of the mutant. In agreement with the finding, subsequent Western blotting and immunohistochemical analysis demonstrated lower levels of DAT protein in the mutant striatum. These results lead to the proposal that, in addition to being a pivotal transcription factor in the immune system, STAT6 may be crucially implicated in mouse behavior by modulating the expression of neuronal genes such as DAT.

Animals↗

PubMed2005

Semaphorin 4A induces growth cone collapse of hippocampal neurons in a Rho/Rho-kinase-dependent manner.

Semaphorins are a family of secreted and membrane-bound proteins, known to control axonal pathfinding. It was recently demonstrated that Semaphorin 4A (Sema4A) is crucially involved in immune cell activation. However, the role of Sema4A in the nervous system has not yet been clarified. To examine if Sema4A can function as a chemo-repulsive cue to growth cones of developing hippocampal neurons, a growth cone collapse assay with recombinant Sema4A was performed in primary hippocampal neurons cultured from E17 mice. In these primary hippocampal neurons, Sema4A induced a significant growth cone collapse as compared with the culture without Sema4A. The Sema4A-induced growth cone collapse could be blocked by Y-27632, a Rho-kinase inhibitor. Furthermore, immunocytochemical analysis with antibodies against Sema4A demonstrated the binding of recombinant Sema4A to the growth cones of hippocampal neurons. Thus, our data indicated that Sema4A could function as a chemo-repulsive cue by activating a receptor whose signal is transmitted to Rho-kinase and induced growth cone collapse of hippocampal neurons.

Animals↗

PubMed2005

Deletion of the kinase domain from death-associated protein kinase attenuates p53 expression in chronic obstructive uropathy.

Death-associated protein kinase (DAPK) is a Ca(2+)/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We previously showed that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) caused by a unilateral ureteral ligation. Here, we used DAPK-mutant mice, generated by the deletion of 74 amino acids from the catalytic kinase domain, to investigate the role of the DAPK kinase domain in regulating the p53 level following COU. The p53 expression levels in obstructed kidneys of wild-type and mutant mice were determined during the course of COU. Western blot analysis revealed that the p53 protein levels were significantly increased at 5 days after a ureteral ligation. This increase in the p53 level was significantly attenuated in mutant kidneys compared to wild-type kidneys. The obstructed kidneys of DAPK-mutant mice showed a significantly lower number of p53-expressing renal tubule cells than wild-type mice. These results are consistent with the hypothesis that DAPK stabilizes p53 protein in response to apoptosis-inducing stimuli. Thus, the present results suggest that the DAPK kinase domain is crucial for stabilizing p53 protein in renal tubular cell apoptosis in a mouse model of COU.

Animals↗

PubMed2005

Reduced radiosensitivity and increased CD40 expression in cyclophosphamide-resistant subclones established from human cervical squamous cell carcinoma cells.

To investigate the interaction between anticancer drug resistance and radioresistance in cervical cancer cells, 3 single cell-derived cyclophosphamide-resistant subclones were established from the drug- and radiosensitive human cervical squamous cell carcinoma cell line ME180 by chronic exposure cultures with 4-hydroxy-cyclophosphamide followed by limiting dilution. The established cyclophosphamide-resistant subclones were also radio- and multidrug-resistant to 7 other anticancer drugs. Flow cytometric analysis revealed significantly increased levels of CD40 expression on the 3 resistant subclones, whereas no CD40 expression was found on the parent ME180 cells. However, there were no changes in the expression levels of CD29, CD49a-CD49f or CD59 between the parent cells and resistant subclones. A recombinant human soluble CD40 ligand had no effect on the proliferation of the resistant subclones. Irradiation had no effect on the 4-hydroxy-cyclophosphamide sensitivity of the parent cells. These results indicate that the established cyclophosphamide-resistant subclones have impaired cell death signals, which are common to both drug- and radiation-induced apoptosis, and cyclophosphamide may not be an adequate drug for use in concurrent chemoradiotherapy. Furthermore, CD40 activation signals may be associated with the multidrug- and radioresistance in these cyclophosphamide-resistant subclones.

Antineoplastic Agents↗

PubMed2005

Radiation-induced cell death is independent of the apoptotic signals mediated by death-associated protein kinase in human cervical squamous cell carcinoma cells.

Death-associated protein kinase (DAPK) was originally identified as a positive mediator of interferon-gamma (IFNgamma)-induced apoptosis in cervical cancer cells, and interferons have been reported to enhance radiosensitivity in various types of squamous cell carcinoma. To examine whether DAPK can regulate cancer cell radiosensitivity, we investigated DAPK expression and radiosensitivity in human cancer cell lines, including the cervical squamous cell carcinoma cell line, ME180, which is both radiosensitive and IFNgamma-sensitive. Of the 5 human cancer cell lines examined, ME180 cells were the most radiosensitive, but their level of DAPK protein expression was undetectable by western blotting. A comparative study of ME180 cells with 2 other uterine cancer cell lines, HHUA and HOKUG, revealed no significant relationships between cellular radiosensitivity and DAPK protein expression or hypermethylation of the DAPK promoter CpG island. INFgamma dose-dependently inhibited ME180 cell proliferation, but did not induce any cell death. IFNgamma significantly enhanced the radiosensitivity of ME180 cells with a slight increase in DAPK protein expression, while irradiation significantly reduced their sensitivity to the growth-inhibitory signals of INFgamma. Analyses of 6 monoclonal cisplatin-resistant subclones established from ME180 cells revealed that all 6 were significantly more radioresistant than the parent ME180 cells without any change in the DAPK protein expression. These results indicate that DAPK does not regulate radiation-induced cell death and that it cannot be either a target molecule for radiotherapy with gene therapy or a prognostic marker for cervical cancer patients treated with radiotherapy.

Antineoplastic Agents↗

PubMed2005

Reduced prepulse inhibition of startle in STAT6-deficient mice.

To determine the role of STAT6 transcription factors in brain function, we performed a battery of mouse behavioral analyses of STAT6-deficient mice. We recently showed that STAT6-deficient mice displayed increased locomotor activities and had reduced levels of dopamine transporter (DAT) expression in the striatum. To further examine the behavioral effects of STAT6 deficiency, we subjected STAT6-deficient mice to behavior task testing prepulse inhibition (PPI) of the auditory startle response. In acoustic prepulse conditions, STAT6-deficient mice displayed significantly lower levels of PPI in acoustic startle than did wild-type mice, indicating their sensorimotor gating deficits. Thus, STAT6 transcription factors may be crucially implicated in sensorimotor gating by modulating the expression of several genes, including DAT in brain neurons.

Acoustic Stimulation↗

PubMed2005

Cell proliferation of bulky cervical squamous cell carcinoma is strongly associated with a paracrine tissue-specific growth factor(s).

To examine the possibility of a cervical squamous cell carcinoma-specific growth factor(s), previously suggested by clinical findings and in vitro culture experiments, we examined in vitro cultures and nude mouse transplantations of cervical squamous cell carcinoma cells obtained from a patient with a bulky cervical tumor without detectable distant metastases. The cancer cells did not proliferate without additional growth factors but remained viable in vitro. Fourteen weeks after transplantation of the tumor cells on their backs, 1 of the 3 nude mice developed large metastatic pelvic tumors without macroscopic metastatic lesions in their lungs or liver. When these pelvic tumor fragments were trans-planted onto the backs of other nude mice, ulcerated back tumors and larger metastatic pelvic tumors, but no macroscopic metastatic lesions in the lungs or liver, were observed. Histopathological examination of these pelvic tumors showed that all were the same squamous cell carcinoma as the primary tumor. These results indicate that cell proliferation of bulky cervical squamous cell carcinoma is strongly associated with a squamous cell carcinoma-specific growth factor(s) in a paracrine manner.

Animals↗

PubMed2005

Combination effects of irradiation and irinotecan on cervical squamous cell carcinoma cells in vitro.

Irinotecan HCl (CPT-11) has frequently been used in chemotherapy or concurrent chemoradiotherapy for patients with advanced cervical cancer, although an effective protocol for chemoradiotherapy with CPT-11 has not yet been established. Using the radiosensitive human cervical squamous cell carcinoma cell line ME180 and SN38, a major active metabolite of CPT-11, both the SN38 effects on radiosensitivity and irradiation effects on SN38 sensitivity were investigated to optimize the chemoradiotherapy protocol for CPT-11. SN38 had no effect on radiosensitivity, and irradiation did not affect SN38 sensitivity. Moreover, 3 of 4 post-irradiation surviving subclones obtained from repeatedly irradiated ME180 cells showed no significant changes in their SN38 sensitivities compared with the non-irradiated parent cells. On the other hand, all 7 SN38-resistant subclones established from ME180 cells showed strong reduction in their radiosensitivities. These results suggest that CPT-11 should be administered to advanced cervical cancer patients after, but not before, standard radiotherapy, and concurrent administration of SN38 with radiotherapy should be avoided in order to prevent severe adverse effects, such as watery diarrhea, which is the main adverse effect of pelvic radiotherapy or CPT-11.

Antineoplastic Agents, Phytogenic↗