Biomedical subjects

Masayuki Takeda

Publications and source records attributed to Masayuki Takeda.

At least 19 recordsLinked to original sources

PubMed2025

Clinical and molecular landscape of metastatic extramammary Paget's disease.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare malignancy without established systemic therapy. EMPD shares molecular features with breast cancer, such as human epidermal growth factor receptor 2 (HER2) and hormone receptor (HR) expression, but their clinical relevance remains unclear. MATERIALS AND METHODS: Tumors from 20 metastatic invasive EMPD cases were analyzed for molecular and biological features. Genomic features, transcriptomic profiles, and HER2 and HR expression status were investigated using immunohistochemistry, fluorescence in situ hybridization, and targeted-genome next-generation sequencing and nCounter BC360 panels. Metastatic breast cancer samples were used as a comparison to clarify metastatic EMPD's clinical relevance. RESULTS: Estrogen receptor expression was observed in 45% of EMPD tumors, while only 10% expressed progesterone receptor. HER2 was overexpressed in 30% of cases, and HER2-directed therapies were durably effective. Among 8 patients with NGS data, 63% (5/8) harbored oncogenic ERBB2 alterations independent of HER2 expression. BC360 profiling revealed biological differences between EMPD and breast cancer, particularly poor biological compatibility for HR-positive tumors. Immune profiling showed that a subset of EMPD tumors exhibited CD8+ T-cell signatures and PD-1/PD-L1 gene expression comparable to triple-negative breast cancer. The median overall survival was 22.1 months (95% CI, 12.0-42.2), with 16 patients (80%) treated with systemic therapy, including anti-HER2 therapy, hormonal therapy, or cytotoxic therapies based on their molecular features. CONCLUSIONS: This study highlights the unique molecular and biological features of metastatic EMPD, emphasizing the need for tailored treatment approaches. This information should be used to guide future clinical strategies for metastatic EMPD.

Humans↗

PubMed2024

Soluble immune checkpoint factors reflect exhaustion of antitumor immunity and response to PD-1 blockade.

BACKGROUNDPrecise stratification of patients with non-small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy.METHODSWe measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay.RESULTSNonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral CD8+ T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors.CONCLUSIONCombinations of soluble immune factors might be able to identify patients unlikely to respond to PD-1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC.TRIAL REGISTRATIONUMIN000019674.FUNDINGThis study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation.

Humans↗

PubMed2006

Gene expression profiling of ATP-binding cassette (ABC) transporters as a predictor of the pathologic response to neoadjuvant chemotherapy in breast cancer patients.

Drug resistance is a major obstacle to the successful chemotherapy. Several ATP-binding cassette (ABC) transporters including ABCB1, ABCC1 and ABCG2 have been known to be important mediators of chemoresistance. Using oligonucleotide microarrays (HG-U133 Plus 2.0; Affymetrix), we analyzed the ABC transporter gene expression profiles in breast cancer patients who underwent sequential weekly paclitaxel/FEC (5-fluorouracil, epirubicin and cyclophosphamide) neoadjuvant chemotherapy. We compared the ABC transporter expression profile between two classes of pretreatment tumor samples divided by the patients' pathological response to neoadjuvant chemotherapy (residual disease [RD] versus pathologic complete response [pCR]) ABCB3, ABCC7 and ABCF2 showed significantly high expression in the pCR. Several ABC transporters including ABCC5, ABCA12, ABCA1 ABCC13, ABCB6 and ABCC11 showed significantly increased expression in the RD (p<0.05). We evaluated the feasibility of developing a multigene predictor model of pathologic response to neoadjuvant chemotherapy using gene expression profiles of ABC transporters. The prediction error was evaluated by leave-one-out cross-validation (LOOCV). A multigene predictor model with the ABC transporters differentially expressed between the two classes (p<or=0.003) showed an average 92.8% of predictive accuracy (95% CI, 88.0-97.4%) with a 93.2% (95% CI, 85.2-100%) positive predictive value for pCR, a 93.6% (95% CI, 87.8-99.4%) negative predictive value, a sensitivity of 88.1%(95% CI, 76.8-99.4%), and a specificity of 95.9% (91.1% CI, 87.8-100%). Our results suggest that several ABC transporters in human breast cancer cells may affect the clinical response to neoadjuvant chemotherapy, and transcriptional profiling of these genes may be useful to predict the pathologic response to sequential weekly paclitaxel/FEC in breast cancer patients.

ATP-Binding Cassette Transporters↗

PubMed2006

Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system.

1. Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. 2. Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE-regulated protein, mitogenesis and cell survival were examined. 3. Exposure of reporter cells to cAMP-elevating agents resulted in time- and concentration-dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8-bromo-cAMP or 8-bromo-cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. 4. Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE-regulated protein, connexin43. 5. Concomitant inhibition of PDE3 and PDE4 attenuated mitogen-induced activation of extracellular signal-regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase-3 and apoptosis. 6. The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells.

1-Methyl-3-isobutylxanthine↗

PubMed2006

ZD6474 inhibits tumor growth and intraperitoneal dissemination in a highly metastatic orthotopic gastric cancer model.

Angiogenesis inhibitors have been used to treat some cancers, but the therapeutic potential of these agents for gastric cancer has remained unclear. To investigate their therapeutic potential, we examined the effect of ZD6474, an agent that selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2; KDR) tyrosine kinase and epidermal growth factor receptor (EGFR) tyrosine kinase, in a highly metastatic orthotopic model using an undifferentiated gastric cancer cell line, 58As1. ZD6474 (100 mg/kg/day, p.o., 2 weeks) significantly inhibited tumor growth (p < 0.05 vs. control) and reduced tumor dissemination into the peritoneal cavity (p < 0.05 vs. control). In addition, to identify putative tumor biomarkers that would reflect the effects of ZD6474 treatment in clinical settings, we examined the gene expression profiles of implanted gastric tumors treated with ZD6474 in vivo. Twenty-eight candidate genes were identified, including IGFBP-3, ADM, ANGPTL4, PLOD2, DSIPI, NDRG1, ENO2, HIG2 and BNIP3L, which are known to be hypoxia-inducible genes. These genes and gene products may be useful biomarkers for monitoring the effects of ZD6474 treatment. ZD6474 also improved the survival of mice with implanted another undifferentiated gastric cancer cell line, 44As3. In conclusion, our results suggest that ZD6474 may have clinical activity against gastric cancer, particularly undifferentiated gastric cancer with peritoneal dissemination. We also identified putative biomarkers for monitoring the pharmacodynamic effects of ZD6474 by gene expression profiling.

Biomarkers, Tumor↗

PubMed2006

Symptom assessment tool for overactive bladder syndrome--overactive bladder symptom score.

OBJECTIVES: Overactive bladder (OAB) is a common symptom syndrome with urgency, urinary frequency, and urgency incontinence. To collectively express OAB symptoms, we developed the overactive bladder symptom score (OABSS). METHODS: Four symptoms--daytime frequency, nighttime frequency, urgency, and urgency incontinence--were scored. The weighing score was based on a secondary analysis of an epidemiologic database. Psychometric properties were examined in five patient groups: OAB (n = 83), asymptomatic controls (n = 34), stress incontinence (n = 29), benign prostatic hyperplasia (n = 28), and other diseases with urinary symptoms (n = 26). RESULTS: The maximal score was defined as 2, 3, 5, and 5 for daytime frequency, nighttime frequency, urgency, and urgency incontinence, respectively. The sum score (OABSS 0 to 15) was significantly greater in the patients with OAB (8.36) than in the other patient groups (1.82 to 5.14). The distribution of the OABSS showed a clear separation between those with OAB and asymptomatic controls. The OABSS correlated positively with the individual scores (Spearman's r = 0.10 to 0.78) and quality-of-life scores assessed by the King's Health Questionnaire (Spearman's r = 0.20 to 0.49). The weighted kappa coefficients were 0.804 to 1.0 for each symptom score and 0.861 for OABSS. The posttreatment reduction in the OABSS was consistent with the global impression of patients of the therapeutic efficacy. CONCLUSIONS: The OABSS, the sum score of four symptoms (daytime frequency, nighttime frequency, urgency, and urgency incontinence), has been developed and validated. OABSS may be a useful tool for research and clinical practice.

Aged↗

PubMed2006

Human muscarinic receptor binding characteristics of antimuscarinic agents to treat overactive bladder.

PURPOSE: We characterized the binding affinities of several antimuscarinic agents in human muscarinic receptors. MATERIALS AND METHODS: Competitive inhibitory effects of antimuscarinic agents on specific NMS [H] (PerkinElmer Life Sciences, Boston, Massachusetts) binding were examined in human tissue homogenates and in CHO-K1 cell membranes expressing human muscarinic receptor subtypes. RESULTS: Oxybutynin, propiverine, tolterodine, the respective metabolites DEOB, DPr-P-4(N-->O) and 5-HM, and darifenacin inhibited in concentration dependent fashion specific [(3)H]NMS binding in homogenates of the human bladder and parotid gland as well as in membranes of CHO-K1 cell lines expressing human muscarinic M(1) to M(5) receptor subtypes. Based on inhibition constant values the inhibitory effects of tolterodine, 5-HM and DPr-P-4(N-->O) were 1.4 to 1.7 times greater in the bladder than in the parotid gland, whereas the inhibitory effects of oxybutynin, DEOB, propiverine and darifenacin were 2 to 10 times greater in the parotid gland. Consequently tolterodine, 5-HM and DPr-P-4(N-->O) compared with oxybutynin, DEOB, propiverine and darifenacin were found to show 3 to 4 times greater affinity to muscarinic receptors in the human bladder than in the parotid gland. Tolterodine and 5-HM were 2-fold more potent for inhibiting specific [(3)H]NMS binding at cell membranes expressing the M(2) vs the M(3) subtype. Conversely oxybutynin, DEOB, propiverine, DPr-P-4(N-->O) and darifenacin showed 2 to 22 times higher affinity to the M(3) than to the M(2) subtype. CONCLUSIONS: Compared with oxybutynin, tolterodine, 5-HM and DPr-P-4(N-->O) may bind more selectively to muscarinic receptors in the human bladder than in the parotid gland.

Humans↗

PubMed2006

Real-time quantitative analysis for human telomerase reverse transcriptase mRNA and human telomerase RNA component mRNA expressions as markers for clinicopathologic parameters in urinary bladder cancer.

AIM: The expression of the telomerase subunits such as human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTR) may be associated with tumor development and progression. We evaluated the relationship between mRNA quantification of both hTERT and hTR and clinicopathologic parameters in bladder cancer. METHODS: We examined the mRNA expression of hTERT and hTR in 29 specimens with bladder cancer (Grade: Grade I, 9 cases; Grade II, 13 cases and Grade III, 7 cases. Stage: pTa-pT1, 18 cases; pT2-T4, 11 cases). We immediately froze all of specimens obtained during TUR-Bt and isolated the total RNA from each specimen. We measured the quantity of hTERT, hTR and GAPDH mRNA by a real-time reverse transcription-polymerase chain reaction method based on TaqMan technology. RESULTS: The hTERT/GAPDH mRNA ratio and hTERT mRNA/total RNA in superficial bladder tumor was significantly lower than in invasive bladder tumor. The hTR/GAPDH mRNA ratio and hTR mRNA/total RNA in superficial tumor were significantly lower than in invasive bladder tumor. The hTERT mRNA expression significantly correlated with tumor grade, but the hTR mRNA expression did not correlate with tumor grade. There was no significant difference in the hTERT/GAPDH mRNA ratio and hTR mRNA/total RNA according to multiplicity of bladder tumor. CONCLUSIONS: Our results demonstrated that the expression of hTERT mRNA correlated with the progression of stage and grade in bladder cancer. The quantitative analysis of hTERT and hTR mRNA might be a marker for clinicopathologic parameters in bladder cancer.

Adult↗

PubMed2006

[Development of the measures to evaluate the similarity of drug brand names].

Brand name confusion is one of the most common causes of drug-related errors. The aim of this study was to develop quantitative measures of similarity among brand names of drugs. We modified the fragmentary pattern-based measure, a measure of similarity for character strings based on the string resemblance system, to develop three novel measures of similarity, i.e., the head and tail-weighted fragmentary pattern-based measure (htfrag), visually weighted htfrag (vwhtfrag), and auditorily weighted htfrag (awhtfrag). The 227 pairs of brand names for which confusion errors have been reported were used as a positive control group. Ten sets of 2270 random pairs of brand names were generated as negative controls. Then we evaluated the measures developed by using the geometric mean of sensitivity and selectivity as an objective function, in comparison with two conventional measures of similarity based on the vector space model (cos1 and htco). The measures developed, htfrag, vwhtfrag, and awhtfrag, provided better discrimination with mean objective function values of 0.953, 0.962, and 0.940, respectively, which were higher than those for the conventional measures cos1 and htco (0.922 and 0.892, respectively). The rates of false-positives and false-negatives were 3.3-10.7% and 5.3-11.9% for cos1, respectively, while the rates for vwhtfrag were 4.8-5.9% and 2.2%, respectively. The measures of similarity developed may provide significant information to avoid drug-related errors associated with brand name confusion.

Medication Errors↗

PubMed2005

Synergistic effects of PDGF-BB and cAMP-elevating agents on expression of connexin43 in mesangial cells.

The gap junction plays an important role in the regulation of cell growth, migration, and differentiation. Platelet-derived growth factor (PDGF) is reported to be a potent inhibitor of gap junctional intercellular communication (GJIC). Short-term exposure of cells to PDGF causes rapid and transient disruption of GJIC without altering connexin43 (Cx43) protein level. In this study, we investigated long-term effects of PDGF-BB on Cx43 expression in mesangial cells (MCs). Exposure of MCs to PDGF-BB affected neither the Cx43 protein level nor GJIC. However, in the presence of cAMP-elevating agents, PDGF-BB dramatically increased the expression of Cx43, which was accompanied by obviously augmented membrane distribution of Cx43 and functional GJIC. The increased expression of Cx43 was closely correlated with reduction in alpha-actin, a dedifferentiation marker of MCs. The effect of PDGF on Cx43 was largely prevented by inhibitors of phosphatidylinositol 3'-kinase or mitogen-activated protein kinase, but not by inhibition of protein kinase C. Exposure of MCs to PDGF-BB caused elevation in intracellular cAMP, and it was abolished by indomethacin, a cyclooxygenase inhibitor. However, indomethacin did not affect the synergistic effect. In addition, PDGF-BB also did not affect the degradation of Cx43. With the use of MCs transfected with a Cx43 promoter-luciferase vector, cooperative activation of Cx43 promoter by PDGF and cAMP was found. Together, our data reveal, for the first time, unexpected synergy between PDGF-BB and cAMP-elevating agents in the induction of Cx43 and MC differentiation. Regulation of GJIC could be an important mechanism via which PDGF modulates MC phenotypes.

Animals↗

PubMed2005

Central nervous system safety of anticholinergic drugs for the treatment of overactive bladder in the elderly.

BACKGROUND: Overactive bladder (OAB) is characterized by urgency and increased frequency of micturition, with or without urinary urge incontinence. Anticholinergic agents are important in the treatment of OAB. However, concerns have emerged about their central nervous system (CNS) safety and the associated risk of cognitive impairment. OBJECTIVE: This article describes the CNS adverse effects of anticholinergic drugs used for the treatment of OAB, with particular emphasis on their use in the elderly. Its objective is to help physicians make optimal choices when selecting anticholinergic treatment for OAB. METHODS: : Relevant data from the literature were identified primarily through a MEDLINE search of articles published through December 2003. The search terms included overactive bladder, central nervous system, anticholinergic, and antimuscarinic. This was not intended to be a systematic review, and articles were chosen for inclusion based on their pertinence to the focus on treatment of OAB in the elderly. RESULTS: Several anticholinergic drugs are available for the treatment of OAB, including oxybutymn, tolterodine, trospium chloride, and propiverine (not available in the United States). Among the agents reviewed, penetration of the blood-brain barrier (as predicted by lipophilicity, polarity, and molecular size and structure) is highest for oxybutymn, lower for tolterodine, and lowest for trospium chloride; limited data are available for propiverine. The total anticholinergic drug burden may also be important in determining the potential for CNS adverse effects. The spectrum of anticholinergic CNS adverse effects ranges from drowsiness to hallucinations, severe cognitive impairment, and even coma. The immediate-release (IR) and extended-release (ER) formulations of oxybutynin have been associated with cognitive impairment. In the only published clinical trial that was identified, no significant differences in CNS adverse effects were observed between the IR and ER formulations of tolterodine. There were few clinical data on the use of propiverine in patients with OAB. Trospium chloride has shown favorable CNS tolerability in postmarketing surveillance studies. CONCLUSION: When considering treatment choices for patients with OAB, particularly the elderly, the potential CNS adverse effects of each anticholinergic agent must be weighed against the severity of OAB symptoms.

Aged↗

PubMed2005

Prevalence and impact on generic quality of life of urinary incontinence in Japanese working women: assessment by ICI questionnaire and SF-36 Health Survey.

OBJECTIVES: To examine the prevalence of urinary incontinence (UI) and the relationship between UI and generic quality of life (QOL) in working women. METHODS: Of 7229 female hospital nurses in Japan, 3734 (51.7%) responded to a self-administered postal questionnaire that included the Rand Medical Outcomes Study 36-item Health Survey (SF-36) and the International Consultation on Incontinence questionnaire (ICIQ) for evaluating symptoms and health-related QOL. RESULTS: The overall prevalence of UI in Japanese working women aged 20 to 64 years (mean 35.2) was 16.7%. The main type of incontinence was stress UI (73%). Its prevalence increased with age and reached a plateau at age 45 to 49 years (34.7%). The ICIQ score also correlated significantly with age. Generic QOL, especially physical functioning, role-physical, social functioning, and role-emotional, was significantly impaired by UI. A comparison of subjects with and without UI revealed that the differences in the physical and mental component summary scores of the SF-36 were largest in the age groups of 25 to 29 and 55 to 59 years, respectively. The correlations of the ICIQ score with the physical and mental component scores were most significant in the age groups of 25 to 29 and 45 to 49 years, respectively. The impact of mixed UI on QOL showed a greater tendency than that of stress or urge UI. CONCLUSIONS: The prevalence of UI showed an age-related increase, with a peak at 45 to 49 years old. The impact of UI on physical health was great in individuals in their 20s, but its impact on mental health increased in the older age groups.

Adult↗

PubMed2005

Continuous, noninvasive monitoring of local microscopic inflammation using a genetically engineered cell-based biosensor.

Using an inflammation-responsive regulatory element as a molecular sensor, we established a cell-based biosensor for continuous, noninvasive monitoring of local microscopic inflammation in vivo. Glomerular mesangial cells were stably transfected with a marker gene encoding secreted alkaline phosphatase (SEAP) under the control of the kappaB enhancer elements. The established cells secreted SEAP in vitro in response to proinflammatory cytokines as well as to soluble factors produced by inflamed glomeruli. To examine feasibility of using the established cells for in vivo monitoring of local microscopic inflammation, the sensor cells were transferred selectively into rat glomeruli via the renal circulation. After induction of acute glomerulonephritis, the serum level of SEAP was increased transiently in cell-transferred nephritic rats. The kinetics of serum SEAP was closely correlated with the natural course of the inflammation, and the increase in SEAP was attenuated by suppression of inflammation using an immunosuppressive drug, cyclophosphamide. Neither cell-transferred normal rats nor nephritic rats without cell transfer exhibited increase in the serum level of SEAP. When the sensor cells were transferred extrarenally, elevation of serum SEAP was not observed in nephritic rats, confirming that the locally settled sensor cells responded only to local inflammation. These results suggested that, without invasive procedures like tissue biopsies, continuous monitoring of microscopic inflammation is feasible in vivo via locally created, cell-based biosensors.

Acute Disease↗

PubMed2005

Modified endoscopic live donor nephrectomy: retroperitoneoscopy followed by hand-assistance.

We have developed a novel modification of previous approaches to donor nephrectomy and herein review our original operative procedure. First, the posterior aspect of the kidney was dissected retroperitoneoscopically and dissection of the renal artery, ureter and gonadal vein was almost completed. Second, the anterior aspect of the kidney was dissected with transperitoneal hand-assistance, and dissection of the renal pedicle from the anterior surface was accomplished easily and safely. This operative procedure was successfully performed for two donors with no intraoperative or postoperative complications. Our modified endoscopic donor nephrectomy is feasible as a minimally invasive procedure because of its safety, and its ability to preserve renal function and establish an excellent operative field for both posterior and anterior aspects of the kidney.

Feasibility Studies↗

PubMed2005

Multifocal metastases of recurrent renal cell carcinoma successfully treated with a combination of low dose interleukin-2, alpha-interferon and radiotherapy.

A 59-year-old man presented with a 2-month history of left flank pain and a possibility of gross hematuria. Left renal cell carcinoma stage II was diagnosed and radical left nephrectomy was performed. Twenty-two months postoperatively, lung metastases were demonstrated and 6 x 10(6) units of alpha-interferon (IFN-alpha) were administered for 9 months, only to keep the sizes of the metastases unchanged. Thirty-four months after the operation, liver metastases and bone metastasis in the left sacroiliac joint were revealed. The combination cytokine therapy was performed with 1.4 x 10(6) U of interleukin-2 (IL-2) and 3 x 10(6) U of IFN-alpha for 16 weeks, and the left sacroiliac joint metastasis was treated with radiation therapy of 4 Gy per day for 7 days. Six months after the 16 weeks of immunotherapy, computed tomography and bone scintigraphy revealed that the metastases of the lung, liver and bone substantially disappeared and this complete response is still kept after 16 months.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Real-time monitoring of mesangial cell-macrophage cross-talk using SEAP in vitro and ex vivo.

BACKGROUND: Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. We established a novel system for continuous, real-time monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. METHODS: Rat mesangial cells were genetically engineered to produce secreted alkaline phosphatase (SEAP) under the control of the nuclear factor-kappaB (NF-kappaB) enhancer elements. The established sensor cells were exposed to macrophages or macrophage-derived factors, and the level of SEAP production was evaluated. RESULTS: In vitro, the established cells expressed and secreted SEAP when exposed to activated macrophages or to cytokines produced by macrophages. The kinetics of SEAP activity in culture media was closely correlated with the expression level of SEAP mRNA. The sensor cells also secreted SEAP in response to media conditioned by macrophage-accumulating, inflamed rat glomeruli. When the sensor cells were transferred adoptively into rat glomeruli subjected to acute anti-Thy 1 glomerulonephritis, the isolated glomeruli containing sensor cells secreted SEAP rapidly and progressively. CONCLUSION: These data suggested that the established system provides simple and useful tools for monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. This approach would be useful for investigation of molecular mechanisms involved in mesangial cell-macrophage interaction and also for screening of therapeutic agents that efficiently interfere with the link between infiltrating leukocytes and resident glomerular cells.

Alkaline Phosphatase↗

PubMed2004

Small in-frame deletion in the epidermal growth factor receptor as a target for ZD6474.

ZD6474 is an inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) tyrosine kinase, with additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. ZD6474 inhibits angiogenesis and growth of a wide range of tumor models in vivo. Gefitinib ("Iressa") is a selective EGFR tyrosine kinase inhibitor that blocks signal transduction pathways implicated in cancer cell proliferation. Here, the ability of gefitinib and ZD6474 to inhibit tumor cell proliferation was examined directly in eight cancer cell lines in vitro, and a strong correlation was noted between the IC(50) values of gefitinib and ZD6474 (r = 0.79). No correlation was observed between the sensitivity to ZD6474 and the level of EGFR or VEGFR expression. The NSCLC cell line PC-9 was seen to be hypersensitive to gefitinib and ZD6474, and a small (15-bp) in-frame deletion of an ATP-binding site (exon 19) in the EGFR was detected (delE746-A750-type deletion). To clarify the involvement of the deletional mutation of EGFR in the cellular sensitivity to ZD6474, we examined the effect of this agent on HEK293 stable transfectants expressing deletional EGFR that designed as the same deletion site observed in PC-9 cells (293-pDelta15). These cells exhibited a 60-fold higher sensitivity to ZD6474 compared with transfectants expressing wild-type EGFR. ZD6474 inhibited the phosphorylation of the mutant EGFR by 10-fold compared with cells with wild-type EGFR. In conclusion, the findings suggested that a small in-frame deletion in the EGFR increased the cellular sensitivity to ZD6474.

Base Sequence↗

PubMed2004

DRESSA: biosensing of dioxin and dioxin-like chemicals using secreted alkaline phosphatase.

In this article, we describe a highly sensitive biosensing system, DRESSA, for detection of dioxin and dioxin-like chemicals. Tandem copies of the dioxin-responsive element (DRE) fused to a minimal viral promoter were subcloned into an expression plasmid upstream of a secreted alkaline phosphatase (SEAP) gene. When murine hepatoma cell line Hepa-1c1c7 was stably transfected with this construct, established sensor clones secreted SEAP following stimulation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A clone HeDS49 was found to be extremely sensitive; it secreted SEAP in response to TCDD in dose- and time-dependent manners, and the minimal detection limit was 100 fM. To detect more than 6 pM of TCDD, the whole assay time (from cell seeding to measurement of SEAP activity) could be reduced to 4h. Secretion of SEAP was induced selectively by other activators of DRE (3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone) but not by activators of unrelated responsive elements. These data suggested that because of the rapidity, easiness, specificity, and high sensitivity of DRESSA, it is more suitable than currently available detection systems for dioxin and dioxin-like chemicals and would be of great advantage to high-throughput screening of these pollutants in environmental samples.

Alkaline Phosphatase↗