IκBNS induces Muc5ac expression in epithelial cells and causes airway hyper-responsiveness in murine asthma models.

BACKGROUND: In allergic asthma, environmental allergens including house dust mite (HDM) trigger pattern recognition receptors and activate downstream signaling pathways including NF-κB pathways not only in immune cells but also in airway epithelial cells. Recent studies have shown that NF-κB activation is regulated positively or negatively depending on the cellular context by IκBNS (encoded by the gene Nfkbid), one of atypical IκB proteins, in the nucleus. Therefore, we hypothesized that IκBNS expressed in immune cells or epithelial cells is involved in the regulation of asthmatic responses. AIM: To determine the roles of IκBNS in HDM-induced asthmatic responses. METHODS: Roles of IκBNS in HDM-induced airway inflammation and airway hyper-responsiveness (AHR) were examined by using IκBNS-deficient (Nfkbid-/- ) mice. Roles of IκBNS expressed in hematopoietic cells and nonhematopoietic cells were separately evaluated by bone marrow chimeric mice. Roles of IκBNS expressed in murine tracheal epithelial cells (mTECs) were examined by air-liquid interface culture. RESULTS: House dust mite-induced airway inflammation and AHR were exacerbated in mice lacking IκBNS in hematopoietic cells. In contrast, HDM-induced airway inflammation was exacerbated, but AHR was attenuated in mice lacking IκBNS in nonhematopoietic cells. The induction of Muc5ac, a representative mucin in asthmatic airways, was reduced in Nfkbid-/- mTEC, whereas the induction of Spdef, a master regulator of goblet cell metaplasia, was not impaired in Nfkbid-/- mTEC. Moreover, IκBNS bound to and activated the MUC5AC distal promoter in epithelial cells. CONCLUSION: IκBNS is involved in inducing Muc5ac expression in lung epithelial cells and causing AHR in HDM-induced asthma models.

MMP13 can be a useful differentiating marker between squamous cell carcinoma and benign hyperkeratotic lesions in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary mechanobullous disease resulting from mutations in the COL7A1 gene, coding for type VII collagen. Patients with RDEB tend to develop squamous cell carcinomas (SCCs) at sites of chronic ulceration or scarring on the whole body. Distinguishing SCC from benign hyperkeratotic lesions is often difficult, not only clinically but also histologically in patients with RDEB. We investigated several matrix metallopeptidase (MMP) subtypes by comparing the DNA amplification microarray findings between evident SCCs and benign hyperkeratotic lesions in the same patient with RDEB. We report that MMP13 was found to be strongly positive in SCCs but negative in benign hyperkeratotic lesions. We found that there is an evident difference in the transitional area between SCCs and benign hyperkeratotic lesions. We propose that MMP13 may be a useful differentiating marker between SCC and benign hyperkeratotic lesions in RDEB.

Induction by estrogen metabolite 16 alpha-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells.

BACKGROUND: Estrogens are potent mammary tumor promoters influencing post-initiation events via epigenetic mechanisms. The upregulation (i.e., induction) of the C16 alpha-hydroxylation pathway during 17 beta-estradiol (E2) biotransformation has been associated with mammary cell transformation. The action of E2 metabolites on tumorigenic transformation, however, is poorly understood. PURPOSE: The newly established mammary epithelial cell line C57/MG, derived from the C57BL mouse strain, was used to examine whether E2 or its metabolites, 16-hydroxyestrone (16 alpha-OHE1) and estriol (E3), function as initiators of mammary cell transformation. METHODS: DNA repair (hydroxyurea-insensitive thymidine uptake), estrogen metabolism (3H exchange to form 3H2O), hyperproliferation (increased cell number), and acquisition of anchorage-independent growth (soft-agar colonies) were used as quantitative end points to measure the relative extent of transformation. RESULTS: Treatment of cells with 200 ng/mL 16 alpha-OHE1 resulted in a 55.2% increase in DNA repair synthesis, a 23.09% increase in proliferative activity, and a 18-fold increase in the number of soft-agar colonies, relative to the solvent controls (P less than .0001). The extent of upregulation of the three end points was similar to that induced by the genotoxic mammary carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA, positive control). DMBA treatment also upregulated the ratio of 16 alpha/C2 hydroxylation of E2 leading to increased formation of 16 alpha-OHE1. E2 and E3 were not effective in upregulating these markers for transformation. CONCLUSION: These results demonstrate that in nontransformed C57/MG cells, 16 alpha-OHE1 may function as an initiator, perturbing the intermediate biomarkers for preneoplastic transformation.

Expression of biomarkers for transformation in 7,12-dimethylbenz[a]anthracene-treated mammary epithelial-cells.

The ability of the mammary procarcinogen 7,12-dimethylbenz(a)anthracene (DMBA) to induce the expression of selected molecular and cellular biomarkers for preneoplastic transformation is examined in a newly developed, immortalized but nontumorigenic mammary epithelial cell line C57/MG. This cell line is established from the mammary tissues of virgin female C57BL/6J strain of mouse. The biomarkers examined included: DMBA-DNA adduct formation and DNA repair (molecular markers), and anchorage-dependent and anchorage-independent growth (cellular markers). Log phase cultures of C57/MG cells were treated for 24 h with 2, 20, and 200 ng/ml of DMBA, and were assayed for DNA adduct formation by the P-32-postlabeling, for DNA repair by the hydroxyurea (HU)-insensitive H-3-thymidine uptake, and for anchorage-dependent and anchorage-independent growth by colony forming efficiency in adherent and non-adherent conditions, respectively. A DMBA dose-dependent increase was detected in DNA adduct formation ranging from 6 adducts/10(9) nucleotides at 2 ng/ml to >1600 adducts/10(9) nucleotides at 200 ng/ml DMBA concentration and in induction of DNA repair synthesis ranging from 10 to 251%. The colony forming efficiency in adherent and non-adherent conditions, exhibited progressive increase up to the dose of 200 ng/ml of DMBA. These results indicate that C57/MG cells are capable of metabolizing the procarcinogen DMBA to generate DNA adducts which may, in part, be responsible for the aberrant proliferation. These molecular and cellular biomarkers that are expressed prior to tumorigenesis may thus constitute useful endpoints for preneoplastic transformation.

Heterogeneous expression of nm23 gene product as a predictor of lymph nodal status in human breast cancer.

The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly low metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between intracellular nm23-immunoreactivity and lymph nodal status of human breast cancer. We analyzed 82 surgically removed breast tumors including 67 invasive carcinomas (ductal, lobular and mucinous carcinomas). The nm23 expression was diffusely positive in the benign tumors and non-invasive carcinomas. Of the invasive ductal carcinomas, lymph node metastasis was found in 67.7% (21/31) of the focally positive/negative cases and in 18.2% (4/22) of the diffusely positive cases (p<0.001). Immunohistochemically, advanced margins of invasive carcinomas with lymph node metastasis were shown to be negative for nm23 expression, while intraductal carcinoma components were positive. This observation suggested that focally positive/negative nm23 expression can be a predictor of lymph node metastasis of invasive ductal breast carcinoma.

Bcl-2 expression in breast cancer is down-regulated by trans-arterial administration of chemotherapeutic agents.

Bcl-2 is one of the cytoplasmic oncoproteins, and has been shown to suppress apoptotic cell death. In this study, we investigated the relationship between Bcl-2 expression and effects of chemotherapeutic agents on human breast cancer cells. We examined 26 surgically resected breast tumors with preoperative trans-arterial administration of chemotherapeutic agents and 30 control cases using immunohistochemical methods. In all 26 cases in the chemotherapy group, the breast cancer cells were focally degenerated to various degrees, associated with inflammation and stromal desmoplastic changes. Bcl-2 expression was found in 46% (12/26) of the chemotherapy group and in 67% (20/30) of controls. Of the 12 Bcl-2-positive cases in the chemotherapy group, 5 were diffusely positive [Bcl-2(2+)] and 7 were focally positive [Bcl-2(+)]. Of the 20 Bcl-2-positive cases in the control group, 18 were diffusely positive and 2 were focally positive. We speculate that Bcl-2 expression was down-regulated by trans-arterial administration of chemotherapeutic agents and was associated with apoptosis and degeneration of breast cancer cells.

Persistent estrogen responsiveness of ras oncogene-transformed mouse mammary epithelial cells.

Ovarian steroids are associated with the proliferation of normal as well as tumorigenically transformed mammary epithelial cells. The experiments performed in this study were designed to establish that (1) tumorigenic transformation induced by the ras oncogene is associated with alterations in estradiol biotransformation, (2) altered endocrine responsiveness persists in the fully transformed tumor cell phenotype and (3) specific perturbations induced by the ras oncogene can be experimentally downregulated. The ras transfectant pH06T and the tumor-derived T1/Pr1 cells exhibited 3- and 43-fold increases, respectively, in C-16 alpha hydroxylation of estradiol relative to the parental mouse mammary epithelial cells (P less than 0.0001). At the cellular level, this alteration corresponded with approximately 90-fold increase in the anchorage-independent growth of T1/Pr1 cells (P less than 0.0001). Estrogen responsiveness of T1/Pr1 cells was demonstrated by their suppression of growth in phenol red-free and/or tamoxifen-supplemented medium and by the reversal of antiproliferative effect of tamoxifen by phenol red and estradiol. Indole-3-carbinol, a naturally occurring tumor suppressive agent, was able to upregulate C-2 hydroxylation at the expense of C-16 alpha hydroxylation of estradiol. Treatment of T1/Pr1 cells with indole-3-carbinol resulted in a substantial decrease in anchorage-independent growth.

Alteration in proliferative and endocrine responsiveness of human mammary carcinoma cells by prototypic tumor-suppressing agents.

The experiments performed in this study were designed to establish that (1) acquisition of anchorage-independent growth, a biological characteristic of tumorigenically transformed phenotype, can be modulated by prototypic tumor-suppressing agents, and (2) modulation of growth is influenced by the metabolic competence of the cells to biotransform estradiol, MCF-7 human breast carcinoma cells exhibited linear cell proliferative kinetics with a 41-hour population doubling time, and a 15% colony-forming efficiency in 0.33% agar. Indole-3-carbinol (13C), a naturally occurring tumor-suppressive agent; tamoxifen (TAM), an antiestrogenic agent; and 4-hydroxytamoxifen (4-OHTAM), a metabolite of TAM, demonstrated 73.7%, 72.5%, and 89.9% suppression in anchorage-independent growth of MCF-7 cells, respectively. At the metabolic level, 13C and 4-OHTAM induced 2.3-fold (P < 0.0001) and 1.3-fold increase (P = 0.001) relative to their own controls in the extent of 2-hydroxylation of estradiol. The results indicate that growth inhibition by 13C, TAM, and 4-OHTAM may in part be due to altered estradiol metabolism in MCF-7 cells. Thus, anchorage-independent growth and altered biotransformation of estradiol may constitute useful cellular and endocrine markers to evaluate the biological response of chemosuppressive agents.

Mechanical model of normal and anomalous diffusion.

The overdamped dynamics of a charged particle driven by an uniform electric field through a random sequence of scatterers in one dimension is investigated. Analytic expressions of the mean velocity and of the velocity power spectrum are presented. These show that above a threshold value of the field normal diffusion is superimposed to ballistic motion. The diffusion constant can be given explicitly. At the threshold field, the transition between conduction and localization is accompanied by an anomalous diffusion. Our results exemplify that, even in the absence of time-dependent stochastic forces, a purely mechanical model equipped with a quenched disorder can exhibit normal as well as anomalous diffusion, the latter emerging as a critical property. Via another interpretation, as the motion of a particle on an inclined rough surface, our results are relevant for the problem of segregation by flow.

Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

First confirmed canine case of Ehrlichia canis infection in Japan.

An 11-year-old castrated Pekinese dog that had been moved from Indonesia to Japan eight years previously was diagnosed with an Ehrlichia canis infection by haematological characteristics (normocytic anaemia, mild thrombocytopenia and hypergammaglobulinaemia) and serological findings (antibody titre to E canis 1:3,200 or more). The dog did not respond to treatment with tetracycline and died from renal failure. The diagnosis was confirmed postmortem by pathological evaluation and polymerase chain reaction (PCR) followed by sequencing of the 16S rRNA gene. Typical morulae of Ehrlichia were detected in the cytoplasm of macrophages in spleen tissue by immunohistological staining. Ehrlichia-like organisms were also detected in the spleen by electron microscopy. E canis-specific PCR analysis of DNA extracted from the spleen gave a positive signal, and sequence analysis of the fragment revealed that it was identical to part of the 16s rRNA gene of E canis. The dog was the first confirmed clinical case of E canis infection in Japan.

[MTT assay using fresh surgical specimens with reference to the transfer system and reproducibility in "test center" method].

A chemosensitivity test (MTT assay) was conducted using 59 fresh surgical specimens collected from Keio University, Kitasato Institute Hospital and 14 affiliated hospitals, in order to assess the specimen transfer system and the reproducibility of the assay results obtained at Keio University and Kitasato Institute Hospital. Although the optical density yielded by the tumor cells in a number of 5 x 10(4)/well and the number of evaluable cases were significantly reduced through the transfer, the chemosensitivity pattern of the specimen was identical before and after the transfer. Fifty seven of 59 cases were evaluable and the concordant rate of the assay results between the two institutes was 80.6% (108/134) among each case-drug combination. Since the transfer system of the specimen was established and the reproducibility of the assay results in two institutes was confirmed, the "test center" method of the MTT assay appears to be possible by collecting the surgical specimens from the affiliated hospitals.

[Enhancement of antitumor activity of mitomycin C against human breast carcinoma xenografts by pretreatment with KM 2210].

Three human breast carcinoma xenografts, MCF-7, R-27 and T-61 serially transplanted into nude mice were treated with mitomycin C (MMC) alone, KM2210 (estra-1, 3, 5(10)-triene-3, 17 beta-diol, 3 benzoate 17-[4-(4-bis(2-chloroethyl)amino)phenyl)-1-oxobutoxy)acetate) alone and KM2210 followed by MMC. One hundred or 300 mg of KM2210 per kg were administered orally daily from Day 1 to 4 and MMC at the dose of 3 mg/kg was given ip on Day 5. The antitumor activity of MMC on these xenografts was enhanced by pretreatment with KM2210, suggesting a new combination chemo- and endocrine therapy of hormone-dependent human breast carcinomas.

[Establishment of transplantable human colon cancer cell lines, chemosensitivity of colon carcinomas and the serially transplantable strains with MTT assay].

Three human colon carcinoma xenografts serially transplantable into nude mice were established and named Co-6, Co-7, and Co-8. The chemosensitivity of these stains were assessed by MTT assay of the fresh surgical specimens (primary MTT assay) and the serially passaged xenografts (xenografts MTT assay), in vivo chemosensitivity test in nude mice (nude mouse system) and clinical responses. Drugs used for the experiments are mitomycin C (MMC), adriamycin (ADM), 5-fluorouracil (5-FU) and cisplatin (DDP). The primary MTT assay revealed true negative with MMC and 5-FU on Co-7 and Co-8 cases. The chemosensitivity of the tumor cells seemed to be increased in the xenografts MTT assay and nude mouse system, in which MMC and DDP were evaluated to be positive on Co-6 and Co-7. However, the chemosensitivity pattern of the tumor cells seemed to be stable in these chemosensitivity tests, indicating better to choose the agent with the highest inhibition rate among various tested agents, even when none have an inhibition rate equal to or more than 50%.

[Asymptomatic pancreatic islet cell tumor (glucagonoma): case report].

A case of asymptomatic pancreatic islet cell tumor (glucagonoma) is reported. A 36-year-old woman undergoing a ultrasonic scan was found to have two masses in the body and tail of the pancreas measuring 5 cm and 4 cm in diameter, respectively. Investigations of serum peptide hormones revealed an elevated glucagon level of 27,500 pg/ml (normal < 100 pg/ml), suggesting the possibility of an islet cell tumor (glucagonoma). The patient, however, was asymptomatic although high levels of glucagon were present. The patient underwent distal pancreatectomy to remove the lesions in the body and tail of the pancreas. Histological findings revealed islet cell tumors of the pancreas, and immuno histochemical staining of the tumor cells demonstrated a positive reaction for chromogranin and glucagon.

Duration of norovirus excretion and the longitudinal course of viral load in norovirus-infected elderly patients.

To prevent dissemination of norovirus in semiclosed environments such as aged-care facilities, it is important to know the period of infectivity in norovirus-infected individuals. We recruited 13 elderly patients aged 60-98 years with norovirus gastroenteritis (11 residents in aged-care facilities and two healthy adults) for this study, and measured the viral loads for norovirus in a total of 63 follow-up faecal samples using a real-time quantitative polymerase chain reaction assay. The average period of norovirus excretion was 14.3 days (range: 9-32 days; median: 13 days). All of the follow-up samples collected between 7 and 10 days after the onset of symptoms tested positive. Viral loads in samples collected between 14 and 18 days after the onset of symptoms were divided into three groups: those testing negative, those with <10(4) copies/g stool, and those with >10(4) copies/g stool. Stools from the group with <10(4) copies/g stool were found to be negative for norovirus up to 21-24 days after the onset of symptoms; however, the group with >10(4) copies/g stool showed prolonged norovirus excretion (up to 32 days) in stools. Although the period of infectivity of excreted viruses has not yet been clarified, these results suggest that careful attention should be taken for at least 14 days after the onset of symptoms and that the measurement of viral load in stools around 16 days after onset might be a useful method for following the course of viral shedding for each patient infected with norovirus.