Noninvasive assessment of regulable transferred-p53 gene expression and evaluation of therapeutic response with FDG-PET in tumor model.

The use of tumor-suppressor gene p53 as an anticancer therapeutic has been vigorously investigated. However, progress has met with limited success to date. Some major drawbacks are the difficulty in achieving controllable and efficient gene transfer as well as in analyzing the transferred gene expression in real time and the treatment response in a timely manner. Thus, development of novel gene transfer vector with a regulative gene expression system coupled with the reporter gene, by which transgene can be monitored simultaneously, is critical. Moreover, noninvasive imaging-based assessment of the therapeutic response to exogenous wild-type p53 gene transfer is crucial for refining treatment protocols. In this study, as a simple preclinical model, we constructed a doxycycline-regulated bidirectional vector harboring a reporter gene encoding red fluorescence protein and p53. Then, we determined the controllable and simultaneously coordinated expression of both proteins and the p53-mediated anticancer effects in vitro and in vivo. Next, we observed that cells or tumors with induced p53 overexpression exhibited decreased uptake of [(14)C]FDG in cellular assay and [(18)F]FDG in positron emission tomography (PET) imaging. Thus, by coupling with bidirectional vector, controllable p53 transfer was achieved and the capability of fluoro-2-deoxy-D-glucose (FDG)-PET to assess the therapeutic response to p53 gene therapy was evidently confirmed, which may have an impact on the improvement of p53 gene therapy.

Nicotine stimulates transcriptional activity of the human dopamine transporter gene.

Nicotine modulates dopaminergic activity in the central nervous system by acting on the reuptake system, including the dopamine transporter (DAT), although precisely remains unclear. Here we investigated the effect of nicotine on the transcriptional regulation of the human DAT (hDAT) gene by conducting luciferase reporter assays. Nicotine enhanced the transcription of hDAT gene constructs in transiently transfected SK-N-SH cells. Hexamethonium, a neuronal (ganglionic) nicotinic acetylcholine receptor antagonist, blocked the action of nicotine. Functional analyses placed the nicotine-responsive region -3.5 to -1.0 kb (from the transcription start site) upstream of the core promotor region. Deletion of intron 1, known as a silencer element of the hDAT gene, abolished nicotine's stimulatory effect. Nicotine failed to stimulate DAT promotor activity in non-neuronal CHO or COS-7 cells or in SK-N-AS cells, another neuronal cell line recently reported as a model for investigating DAT gene expression. These results suggest a nicotinic cholinergic mechanism to be involved in the nicotine-induced up-regulation of DAT gene expression.

5-Methoxy-N,N-diisopropyltryptamine (Foxy), a selective and high affinity inhibitor of serotonin transporter.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a synthetic orally active hallucinogenic tryptamine derivative, known also as Foxy or Foxy methoxy. However, few studies have examined its effects in vitro. In the present study, we investigated the actions of 5-MeO-DIPT against monoamine neurotransmitter transporters, including the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using COS-7 cells heterologously expressing these transporters and rat brain synaptosomes. 5-MeO-DIPT specifically inhibited the uptake of [3H]serotonin (5-HT) by the SERT-expressing COS-7 cells and rat striatal synaptosomes in a high affinity manner at concentrations similar to those for cocaine. The effect was reversible and competitive. 5-MeO-DIPT failed to stimulate reverse transport of [3H]5-HT through SERT, while it prevented the releasing action of methamphetamine. 5-MeO-DIPT induced cell toxicity at high concentrations in COS-7 cells, and it was not influenced by the expression of SERT. These results demonstrated that 5-MeO-DIPT acts as a competitive SERT inhibitor and has an inability to cause reverse transport, underlying its serotonergic actions.

The changes of hepatic metallothionein synthesis and the hepatic damage induced by starvation in mice.

Metallothionein (MT) is induced in the liver not only by heavy metals, but also by stress such as starvation. However, the meaning of the induced MT during starvation has never been clear. In this study, we investigated the relationship between changes in hepatic MT synthesis and the hepatic damage that occurs during starvation. MT synthesis was assessed by measuring MT contents and the expression of the MT gene in the liver. The hepatic damage was assessed by measuring glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) activities in the serum. MT synthesis in the liver increased over the normal level by starvation, but decreased under the normal level by refeeding after starvation. Both GPT and GOT activities of the refeeding group were higher than those of the control group. However, MT synthesis increased by a subcutaneous injection with CdCl(2) (1 mg Cd /kg) at the same time as refeeding after starvation. At this point, GOT activity decreased until it reached the normal level. MT synthesis decreased by refeeding after starvation, and from the results found in this study, we proposed the hypothesis that the liver damage caused by refeeding after starvation might be due to the decrease in the synthesis of a sufficient amount of MT induced by metals.

Metallothionein expression and localization in rat bone tissue after cadmium injection.

We investigated the induction of metallothionein (MT) by cadmium (Cd) in the bone tissue of rats. To clarify the cell response to Cd in bone, the isoform-specific expression of MT mRNAs (MT-I and MT-II) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Both MT-I and MT-II mRNA levels were increased within 3 h by Cd administration. MT (MT-I/MT-II) localization after single Cd injection were also confirmed by immunohistochemical studies. Notably, MT-positive cells were time-dependently increased, and the positive cells were mainly localized in osteocytes. The cell-specific induction of MT may be associated with Cd accumulation and Cd-induced bone injury in vivo. Furthermore, we also found that MT was consecutively expressed in some osteoclasts of control rats. This finding suggested a new role of osteoclasts in bone metabolism.

Reduction of hematopoietic cell-specific tyrosine phosphatase SHP-1 gene expression in natural killer cell lymphoma and various types of lymphomas/leukemias : combination analysis with cDNA expression array and tissue microarray.

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

Localization of metallothionein (MT) and expression of MT isoforms induced by cadmium in rat dental pulp.

We investigated the induction of metallothionein (MT) by cadmium (Cd) in the dental pulp of rat incisors. Time-course studies of MT mRNA expression after single Cd injection were observed by Northern-blot analysis. The isoform-specific expressions of MT mRNAs (MT-I, MT-II and MT-III) were observed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Both MT-I and MT-II mRNA levels increased within 3 h, peaked at 3 h and then decreased. These findings demonstrated that MT-I and MT-II mRNA were rapidly induced by Cd in dental pulp. MT-III mRNA was constitutively expressed in rat dental pulp, but the expression level did not change by Cd treatment. The localization of MT protein in Cd-treated rat dental pulp was determined by immunohistochemical staining using anti-MT antibody against MT-I and MT-II. MT protein was localized in the specific cell type of odontoblasts (secretory odontoblasts and resting odontoblasts). In conclusion, it is likely that stained MT in the immunohistochemical study should be MT-I and/or MT-II. Furthermore, MT-I and/or MT-II in Cd-treated rat dental pulp was localized in odontoblasts, in which accumulation of Cd were reported. The cell-specific synthesis of MT may be associated with its metal storage and detoxification role in dental tissues.

The effects of ovariectomy and female sex hormones on hepatic metallothionein-I gene expression after injection of cadmium chloride in mice.

Metallothioneins (MTs) have a low molecular weight and have been considered to be important metal-binding proteins in defense from cadmium (Cd) toxicity in animals. These proteins are known to be induced by the injection of heavy metals such as Cd. Previously, we developed the measurement of the MT mRNA expression by the RT-PCR method. In this study, to clarify the relation between Cd-induced MT-I mRNA expression and female sex hormones in liver, we investigated the influences of the ovariectomy and female sex hormones on hepatic MT-I mRNA expression after Cd injection, and also investigated the effects of aging on hepatic MT-I mRNA expression in mice after Cd injection. We analysed the MT-I mRNA expression by the RT-PCR method. Cd-induced MT-I mRNA expression in ovariectomized mice was more than that in sham-operated mice (9 weeks old). Both 17 beta -estradiol and progesterone reduced the Cd-induced MT-I mRNA expression in ovariectomized mice (9 weeks old). Moreover, the MT-I mRNA expression in male mice was more than that of females (9 weeks old). However, the sex difference in the gene expression was not found in younger (4 weeks old) or older (46 weeks old) mice. These results suggest that the expression of hepatic MT-I mRNA after Cd injection is influenced by female sex hormones.

Opposing pharmacological actions of cepharanthin on lipopolysaccharide-induced histidine decarboxylase activity in mice spleens.

A biscoclaurin alkaloid preparation, cepharanthin (Ceph), is reported to have opposing pharmacological effects, enhancement or depression, on several cells and tissues, although detailed mechanisms remain unclear. Previously, we reported that Ceph enhanced lipopolysaccharide (LPS)-induced histidine decarboxylase (HDC) activity in mice spleens by consecutive pre-administration. In this study, we examined the pharmacological effects on HDC activity of a single Ceph pre-administration to test the influence of the administration method. Consequently, HDC activities were decreased by a single administration 15 minutes before LPS challenge in ddY and ICR mice spleens. Moreover, to further examine this suppressing effect, we employed genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice to avoid the influence of mast cells. In W/Wv mice, HDC activity was enhanced, but not in the congenic WBB6F1 +/+ mice. These findings suggest that mast cells influence the depressant effect on HDC activity by a Ceph single administration in mast cell sufficient mice.

Alteration of odontoblast osteonectin expression following dental cavity preparation.

Cavity preparation can increase the active synthesis and secretion of non-collagenous proteins by odontoblasts, thus resulting in the deposition of tertiary dentine. In this study, the effect of cavity preparation on osteonectin expression was examined in odontoblasts of the rat tooth pulp. A class V cavity was prepared in rat first molars to stimulate odontoblastic secretory activity, and the animals were killed at various intervals. In the normal pulp, osteonectin immunoreactivity was detected in odontoblasts but not other cells. At 1 day after cavity preparation, immunoreactivity had diminished beneath the cavity. At 3 days, strong immunoreactivity could be detected in odontoblasts beneath the cavity. Numerous round cells underlying the odontoblastic layer also demonstrated immunoreactivity. Thereafter, the intensity of osteonectin immunoreactivity in odontoblasts beneath tertiary dentine decreased gradually, and at 30 and 60 days, it was weaker than in normal pulp. These findings suggest that osteonectin is actively synthesized by odontoblasts underlying a cavity in the initial stage of tertiary dentine formation.

Localization, regulation, and function of metallothionein-III/growth inhibitory factor in the brain.

The metallothionein (MT) family is a class of low molecular, intracellular, and cysteine-rich proteins with a high affinity for metals. Although the first of these proteins was discovered nearly 40 years ago, their functional significance remains obscure. Four major isoforms (MT-I, MT-II, MT-III, and MT-IV) have been identified in mammals. MT-I and MT-II are ubiquitously expressed in various organs including the brain, while expression of MT-III and MT-IV is restricted in specific organs. MT-III was detected predominantly in the brain, and characterized as a central nervous system-specific isomer. The role of MTs in the central nervous system has become an intense focus of scientific research. An isomer of MTs, MT-III, of particular interest, was originally discovered as a growth inhibitory factor, and has been found to be markedly reduced in the brain of patients with Alzheimer's disease and several other neurodegenerative diseases. MT-III fulfills unique biological roles in homeostasis of the central nervous system and in the etiology of neuropathological disorders.

Bisphenol A enhances cadmium toxicity through estrogen receptor.

To clarify the action of estrogenic endocrine disruptors on cadmium (Cd)-induced metallothionein (MT) synthesis in the liver, we investigated the effects of bisphenol A (BPA) on hepatic MT-I mRNA expression and MT contents after Cd injection. Liver damage after Cd injection was assessed by measuring glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) activities in the serum. It was found that BPA reduced the Cd-induced expression of MT-I mRNA and MT protein in the liver. The administration of tamoxifen, an estrogen receptor antagonist, prevented the reduction of hepatic MT content by PA. Moreover both the GPT and GOT activities of the BPA-treated groups were higher than those of the control groups. These findings suggest that BPA reduced hepatic MT synthesis after Cd injection via the estrogen receptor which resulted in increased damage to the liver.

Expression of metallothionein-III mRNA and its regulation by levodopa in the basal ganglia of hemi-parkinsonian rats.

In the brain, metallothionein (MT)-III exhibits a free radical scavenging activity. Here we examined the expression of MT-III mRNA in the basal ganglia of 6-hydroxydopamine (6-OHDA)-lesioned hemi-parkinsonian rats and its regulation by levodopa. The level of MT-III mRNA was significantly decreased in the striatum of 6-OHDA-lesioned side. Levodopa treatment significantly increased the expression of striatal MT-III mRNA in the non-lesioned side, but showed no significant effect in the 6-OHDA-lesioned side. These results suggest that the regulation of MT-III mRNA may be related to the progressive degeneration in parkinsonism.

Antioxidants protect against dopamine-induced metallothionein-III (GIF) mRNA expression in mouse glial cell line (VR-2g).

Metallothionein (MT)-III, originally discovered as a growth inhibitory factor (GIF), is a brain specific isomer of MTs and is markedly reduced in the brain of patients with Alzheimer's disease (AD) or other neurodegenerative diseases. We analyzed the level and regulation of mRNA expression of MT-III in immortalized fetal mouse brain glial cells (VR-2g) by reverse transcriptase-polymerase chain reaction (RT-PCR). We have recently reported that dopamine (DA) increases the expression of MT-III mRNA in vitro. In this study, we investigated the mechanism of such increase by examining the effects of DA agonists (SKF38393 or bromocriptine) and DA antagonists (SCH23390 or sulpiride) on the expression of MT-III mRNA. MT-III mRNA did not change by either agonist and DA-increased MT-III mRNA was not inhibited by either antagonist. These results suggested that the induction of MT-III mRNA by DA was not mediated by stimulation of DA receptors. On the other hand, DA-induced MT-III mRNA expression was strongly inhibited by the addition of antioxidants (glutathione, vitamin E or ascorbic acid), indicating that DA-enhanced MT-III mRNA was mediated by reactive oxygen species. Our results suggest that oxidative stress may be one of the principle factors that modulate MT-III mRNA expression.

[The 1999 domestic state of development of anti-asthma].

According to the "Guideline for Diagnosis and Treatment of Asthma" established by the Japanese Society of Allergy in 1998, inhaled adrenergic beta 2 agonists and inhaled corticosteroids are recommended for treatment of asthma. Thereafter, the development of new drugs for the treatment of asthma has begun changing. The concepts upon which the development of investigational drugs for asthma are based include improvements of drug delivery systems (ease of use, long-acting preparations, fewer side-effects), device design and appropriate auxiliary instrumentation. Moreover, chronic asthma has come to be recognized as an inflammatory disease of airway mucosa. At present, various antiallergic compounds such as tachykinin, leucotrien, PAF antagonists and others are under investigation thanks to the identification of new chemical mediators of airway inflammation and studies have progressed to the synthesis and manufacturing of new pharmaceuticals with antagonistic action. Thus, this review classified and introduced various new investigational anti-asthma and further describes the structure-activity relationships of beta 2 agonists and inhaled corticosteroids.

Induction of metallothionein mRNA expression in the mouse liver after cadmium injection as measured by the reverse transcriptase-polymerase chain reaction method.

Metallothioneins (MTs) are low molecular weight proteins that have been considered to be important metal-binding proteins in the defense against cadmium (Cd) toxicity in animals. These proteins are known to be induced by the injection of heavy metals such as Cd. Previously, we developed the RT-PCR method to measure the level of MT mRNA expression. In this study, we analyzed the time course and dose response of Cd-induced MT-I mRNA expression in the male and female mouse liver. By this method, we measured hepatic MT-I mRNA expression which reached the highest peak 6 h after subcutaneous injection of 1.1 mg/kg Cd in 9-week old male and female mice compared with those of corresponding controls (0 h). There was no statistical difference in the hepatic MT-I mRNA expression between male and female mice 6 h after the injection of this dosage. However, it is notable that the MT-I mRNA expression in male mice was much higher than that in females 6 h after injection of 0.5 mg/kg Cd. Thus, the induction of hepatic MT-I mRNA expression is dependent on the sex of the mouse, the dosage and the time course of Cd.

Cytotoxicity of a trial resin composite liner containing TiK2F6 on rat dental pulp cells.

The aim of this study was to assess the toxicological responses of a resin composite containing TiK2F6 and NaF in rat dental pulp cells. Trial resin composite liners were made, containing 3 wt% fluorides (TiK2F6 or NaF). These specimens were immersed in 5 ml of cell culture medium supplemented at 37 degrees C for 24 hours. The eluates were used for the experiments. We judged the cytotoxicity of the samples by the cell viability. The original elute solution was serially diluted and then the medium was exchanged for the dilute medium. The cell viability at 1, 2 or 5 days after commencement of re-culturing was calculated. The viability of cells in the eluate from the resin composite liners containing TiK2F6 and NaF decreased with time. The cytotoxicity of TiK2F6 was weaker than that of NaF at all times.