Induction of indistinguishable gene expression patterns in rats by Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.

Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats.

Constitutive activation of nuclear factor-kappaB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines.

Constitutive nuclear factor (NF)-kappaB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kappaB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kappaB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kappaB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-kappaB activation by expression of IkappaBalpha super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-kappaB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-kappaB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-kappaB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer.

Behavior in the forced swim test and neurochemical changes in the hippocampus in young rats after 2-week zinc deprivation.

Abnormal behavior in zinc deficiency and its cause are poorly understood. In the present paper, behavior in the forced swim test and neurochemical changes in the brain associated with its behavior were studied focused on abnormal corticosterone secretion in zinc deficiency. The effect of chronic corticosterone treatment was also studied. Immobility time in the forced swim test was increased in young rats fed a zinc-deficient diet for 2 weeks, as well as corticosterone (40mg/kg/dayx14 days)-treated control rats. The basal Ca(2+) levels in the hippocampus, which were determined by fluo-4FF, AM, were increased in both brain slices from zinc-deficient and corticosterone-treated rats. Serum glucose level was decreased in zinc deficiency and hippocampal glucose metabolism, which is determined by [(14)C]2-deoxyglucose uptake, was elevated. Hippocampal ATP level was not decreased, whereas, the concentrations of glutamate, GABA and glutamine in the hippocampus, unlike the whole brain, were decreased in zinc deficiency. However, the decrease in these amino acids was restored by adrenalectomy prior to zinc deficiency. These results suggest that glucose is insufficient for the synthesis of amino acids in the hippocampus of zinc-deficient rats. It is likely that the neurochemical and metabolic changes in the hippocampus, which may be associated with abnormal corticosterone secretion, is the base of abnormal behavior associated with neuropsychological symptoms in zinc deficiency.

Application of quantitative gene expression analysis for pertussis vaccine safety control.

Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.

Application of DNA microarray technology to influenza A/Vietnam/1194/2004 (H5N1) vaccine safety evaluation.

We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.

NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells.

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency.

On the basis of abnormal neuropsychological behavior in the open-field test after 2-week zinc deprivation, neurochemical response was examined in young mice fed a zinc-deficient diet for 2 weeks. Serum corticosterone concentration was markedly higher in zinc-deficient mice than in the control mice. Basal signals of intracellular calcium (fluo-4 FF) were also significantly more in hippocampal slices from zinc-deficient mice. These results suggest that basal Ca2+ levels in hippocampal cells are increased by zinc deficiency. On the other hand, Schaffer collateral long-term potentiation (LTP) was unaffected by zinc deficiency; the averaged fEPSP after tetanic stimulation was 162+/-8% of baseline value in the control and 172+/-22% in zinc-deficient mice. In the Morris water maze, there was also no significant difference in learning behavior for the hidden platform task between the control and zinc-deficient mice. The present study indicates that Schaffer collateral LTP associated with spatial cognition performance are unaffected by calcium dyshomeostasis in the hippocampus elicited by 2-week zinc deprivation, which may be linked to the increased serum corticosterone concentration.

FoxA1 as a lineage-specific oncogene in luminal type breast cancer.

The forkhead transcription factor FoxA1 is thought to be involved in mammary tumorigenesis. However, the precise role of FoxA1 in breast cancer development is controversial. We examined expression of FoxA1 in 35 human breast cancer cell lines and compared it with that of ErbB2, a marker of poor prognosis in breast cancer. We found that FoxA1 is expressed at high levels in all ErbB2-positive cell lines and a subset of ErbB2-negative cell lines. Down-regulation of FoxA1 by RNA interference significantly suppressed proliferation of ErbB2-negative and FoxA1-positive breast cancer cell lines. Down-regulation of FoxA1 also enhanced the toxic effect of Herceptin on ErbB2-positive cell lines through induction of apoptosis. Taken together with previous data that FoxA1 is a marker of luminal cells in mammary gland, our present results suggest that FoxA1 plays an important role as a lineage-specific oncogene in proliferation of cancer cells derived from mammary luminal cells.

Two vaccine toxicity-related genes Agp and Hpx could prove useful for pertussis vaccine safety control.

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.

Influence of inhalation anesthesia assessed by comprehensive gene expression profiling.

Although general anesthesia is routinely used as an essential surgical procedure and its harmlessness has been evaluated and endorsed by clinical outcomes, little is known about its comprehensive influence that is not reflected in mortality and morbidity. In this paper, we have shown that inhalation anesthesia affected the expression of <1.5% of >10,000 genes, by analyzing the expression profiles for multiple organs of rats anesthetized with sevoflurane. The small number of transcripts affected by the inhalation anesthesia comprised those specific to single and common in multiple organs. The former included genes mainly associated with drug metabolism in the liver and influenced by agents such as amphetamine in the brain. The latter contained multiple circadian genes. In the brain, we failed to detect the alteration of the clock gene expression with the exception of Per2, assuming that anesthesia perturbs circadian rhythms. Our findings provide the first assessment for the influence of inhalation anesthesia by approaches of experimental biology and genome science.