Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

Protection by cycloheximide against cytotoxicity induced by vincristine, colchicine, or delta 12-prostaglandin J2 on human osteosarcoma cells.

We examined the protective effects of cycloheximide against cytotoxicity induced by vincristine, colchicine, delta 12-prostaglandin J2, or other antitumor agents on the human osteosarcoma cell line, KSu. Vincristine at a concentration of 0.5 microgram/ml decreased the initial cell number to 34% during 4 days; however, when cycloheximide (0.5 to 10 micrograms/ml) was coexistent, the decrease of the cell number was suppressed and 68% of the initial cells remained viable at the maximum. Furthermore, 0.1 micrograms/ml of cycloheximide also reduced cytotoxicity of colchicine (0.1 to 5 microM) or delta 12-prostaglandin J2 (1 to 5 micrograms/ml) and reduced the cytotoxicity of 0.1 microgram/ml of doxorubicin or 1 micrograms/ml of mitomycin C, suggesting that protection by cycloheximide is shown against cytotoxicity of various types of antitumor agents even on human malignant cells. Next, protein synthesis was reduced to 52% of a control at 3 h by 0.1 micrograms/ml of cycloheximide, suggesting that protein synthesis inhibition precedes the protection. De novo protein synthesis analysis showed that vincristine (0.5 microgram/ml) does not induce any specific protein, whereas delta 12-prostaglandin J2 (3 or 4 micrograms/ml) induced Mr 70,000 and 90,000 proteins, and these were markedly inhibited by cycloheximide (0.1 microgram/ml). In a cell-cycle study, M-phase arrest by vincristine (0.5 microgram/ml) was inhibited in the presence of 0.1 microgram/ml of cycloheximide, suggesting that cell cycle arrest by cycloheximide may be important for protection. From these data, this protection by cycloheximide seems to be more general than expected before.

Genistein arrests cell cycle progression at G2-M.

Genistein, an isoflavone, is a specific inhibitor of tyrosine kinase and topoisomerase II. However, its effect on cell growth is unknown. Therefore, we examined the effects of genistein on cell growth and cell cycle progression and compared its effects with other flavonoids. Genistein inhibited in a dose-dependent manner the growth of HGC-27 cells derived from human gastric cancer. Flow-cytometric analysis showed that genistein almost completely arrested the cell cycle progression at G2-M. This effect was reversible when genistein was removed from the culture medium. In contrast, other flavonoids such as flavone, luteolin, and the structurally similar daidzein arrested the cell cycle at G1. Consistent with the flow-cytometric analysis, microscopic observation showed that genistein did not increase the mitotic index, which supposes that genistein may arrest the cell cycle at G2 or early M. These results suggest that the G2-M arrest by genistein is a unique effect among flavonoids.

CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene.

Cytosine methylation of CpG sites in the promoter region of eucaryotic genes is involved in the inactivation of expression of certain genes. Given that methylation can lead to reduced transcription, it is possible that expression of tumor-suppressor genes is also inactivated by hypermethylation, thereby contributing to the etiology of cancer. Recently we found five sporadic retinoblastoma tumors (16% of all unilateral cases) with hypermethylation of the 5' end of the retinoblastoma gene without detecting any structural abnormalities. However, it is unclear whether the promoter of the retinoblastoma gene is actually inactivated by its hypermethylation. Here we show that specific hypermethylation in the promoter region of the retinoblastoma gene reduces its expression to only 8% of the unmethylated control. Furthermore, we have found that two transcription factors important for the promoter activity, an activating transcription factor (ATF)-like factor and the retinoblastoma binding factor 1, do not bind when their recognition sequences are CpG methylated. These results in vitro strongly support the hypothesis that CpG methylation of the human tumor-suppressor gene can result in the inactivation of the gene and thus lead to oncogenesis.

N-myc suppression and cell cycle arrest at G1 phase by prostaglandins.

Effects of cyclopentenone prostaglandins, delta 12-prostaglandin (PG) J2 and PGA2 on the expression of N-myc in relation to the effects on cell cycle progression were investigated using human neuroblastoma cell line GOTO. Both PGs suppressed N-myc expression within several hours prior to inducing G1 arrest. The N-myc suppression with delta 12-PGJ2 was continued but with PGA2 it was gradually released, followed by the release of G1 arrest. These results suggest that delta 12-PGJ2 and PGA2 inhibit cell cycle progression in strong association with N-myc suppression and delta 12-PGJ2 is more potent and has a longer effect than PGA2.

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells.

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.

Age-related enhancement of tumor necrosis factor (TNF) production in mice.

We investigated age-related changes in the production of TNF at the cellular level using immunocompetent peritoneal and spleen cells from C3H/He mice of various ages. The density of cultured peritoneal macrophages and spleen cells required for TNF production was at least 5 x 10(5) cells/dish. The optimal concentration of OK-432 for 24-h culture of peritoneal macrophages (1 x 10(6) cells) and spleen cells (1 x 10(7) cells) was 0.5 and 0.1 KE/ml, respectively. Among peritoneal cells, adherent macrophages were the major TNF-producing cells, whilst nonadherent T or B cells alone did not produce TNF after stimulation with OK-432. In the case of spleen cells, T or B cells were involved in the production of TNF when cultured with a few adherent cells in the presence of OK-432. However, T or B cells alone failed to produce TNF. Production of TNF by peritoneal macrophages from both male and female mice increased significantly with aging. In contrast, although TNF production by spleen cells tended to increase with aging, no significant change was noted. The total number of peritoneal and spleen cells, respectively increased up to about 18 months after birth with B cells being principally responsible for this age-related increase. We previously reported that systemic production of TNF increases with aging. The present study of TNF production at the cellular level in mice indicated (1) that TNF production per macrophage increased with aging, and (2) that the number of T and B cells involved in the production of TNF in the presence of macrophages also increased at least up to middle age.

Strain differences of age-dependent changes in the responsiveness to a T-independent type-2 antigen in mice.

We have assessed age-associated early changes in antibody response to a T-independent type-2 (TI-2) antigen, dinitrophenylated ficoll (DNP-Ficoll). Mice of most strains, including long-lived and autoimmune-prone strains, give a high response when approximately 2 months old; thereafter the response declines sharply to the 3rd-4th month of age and continues to do so, more gradually, up to the age of 6 months. Age-related changes in the response of C57BL/6 mice follows a different course: the response remains unchanged up to the first year of life, i.e. to middle age. The in vitro anti-DNP-Ficoll antibody response of B cells could be increased by the addition of young syngeneic T cells. The augmenting activity of splenic T cells of C3H/He mice declines clearly as a function of age. In contrast, splenic T cells of C57BL/6 mice have low augmenting activity whether the T cells are obtained from young or middle-aged donors. Unlike the augmenting capacity of T cells, B-cell responsiveness to DNP-Ficoll increases until middle age in all strains examined. We conclude that early age-associated changes in antibody response to the TI-2 antigen is polymorphic and that the early age-related decline in in vivo responsiveness is attributable to an age-associated decline in augmenting T helper cell activity.

Studies on age-related functional changes in regulatory T cells and B cells involved in the autoantibody production of MRL/MpJ- +/+ mice.

Age-related changes in anti-DNA autoantibody production of MRL/MpJ- +/+ mice were investigated. In lipopolysaccharide (LPS)-stimulated cultures, spleen cells of the mice showed an age-related, marked increase in the ability to produce IgG class of the autoantibody after the age of 12 months, while they showed a tendency to decrease with age in the production of IgM class of the autoantibody. Serum levels of anti-DNA autoantibodies rose markedly in the IgG autoantibody but not in the IgM autoantibody after 12 months of age, which is well consistent with the observation in the LPS-stimulated cultures. T cell-depleted spleen cells, however, showed only a small increase with age in the IgG autoantibody productive ability. These results suggest that the age-associated increase in the IgG autoantibody production in the mice is under T-cell control. Age-associated changes in suppressor capacity in spleen cells of the mice were also investigated. Suppressive activity of the cells stimulated by 2-day incubation with concanavalin A (Con A) showed a clear increase as the donor age advanced, when assayed on the LPS-stimulated anti-DNA autoantibody production in vitro. The results indicate that, in MRL/MpJ-+/+ mice, suppressor capacity does not decline with age and is not related as a cause to the autoantibody production.

The effect of taurine on age-related immune decline in mice: the effect of taurine on T cell and B cell proliferative response under costimulation with ionomycin and phorbol myristate acetate.

Proliferative responses to the costimulation with phorbol-12-myristate-13-acetate (PMA) and suboptimal doses of ionomycin in the purified T and B cells from old mice were lower than those from young mice. The degree of the age-related decline was more significant in T cells than in B cells. Taurine, a sulfur containing amino acid, augmented the proliferative responses of T cells from both young and old mice. The augmentation of the proliferative response by taurine was more marked in old T cells than in young ones. The concentration of intracellular free calcium ion ([Ca2+]i) was significantly lower in the old T cells under the stimulation with PMA and ionomycin than that in the young ones. In the presence of taurine, the concentration of [Ca2+]i in the old T cell significantly increased under the stimulation. The results indicate that taurine improved the proliferative response of old T cells by the restoration of the increment of the concentration of [Ca2+]i under the stimulation.

Effect of food-restriction stress on immune response in mice.

Daily 23-h food deprivation for 1-5 days induced gastric ulcers and atrophic changes of the spleen and thymus, accompanied by a rise in plasma cortisol and catecholamine levels in mice. It also modulated several immune cell functions in the spleen including a drop in the B cell population but no change in the mitogen response of the B cells, an increase in T cell population but no change in the L3T4/Lyt2 ratio and an early increase in natural killer activity and O2- production by macrophages. These effects are thought to correlate to the increase in stress-associated humoral factors and this may partly result from stress induced by food restriction.

Aging effect on the immune functions of murine gut-associated lymphoid tissues.

Immune functions generally decline with aging. However, the onset and the rate of the functional decline may be different in each lymphoid compartment. We studied the effect of aging on the murine Peyer's patch (PP) cells, and mesenteric lymph node (MLN) cells, which are a part of gut-associated lymphoid tissues (GALT). The capacity of proliferative responses to mitogens of lymphoid cells from GALT decreased with aging. However, the rate of the decrease was much slower than that in the spleen cells. Production of interleukin-2 (IL-2) and interleukin-3 (IL-3) in aged T cells was also studied. IL-2 production of T cells from PP, MLN, spleen cells decreased with age. The age-related decrease was observed at 21 months of age in spleen and at 24 months of age in PP and MLN cells. In contrast, IL-3 production of PP, MLN, spleen cells didn't decrease at 21 months of age, but decreased only in spleen cells at 24 months of age. Therefore, it is suggested that the onset and the rate of age-related functional decline of GALT are much later and slower than those of systemic immune system. GALT seems to maintain the immune functions longer than systemic immune system.

Effect of ammonia on cell-cycle progression of human gastric cancer cells.

AIM: Ammonia is a cytotoxic factor of Helicobacter pylori that is involved in gastric mucosal injury. This study was designed to show whether ammonia has an effect on the cell-cycle progression in human gastric cells in vitro. MATERIALS AND METHODS: We studied the effects of ammonia and ammonium chloride on cell growth and cell-cycle progression of the human gastric cancer cell line HGC-27. We cultured HGC-27 cells and counted viable cells by trypan blue dye exclusion 24 h after the addition of various concentrations of ammonia or ammonium chloride. DNA contents of nuclei were analysed by flow-cytometry. RESULTS: Ammonia and ammonium chloride inhibited the proliferation of HGC-27 cells dose-dependently. Flow-cytometric analysis showed S-phase accumulation of HGC-27 cells treated with ammonia and ammonium chloride at cytostatic doses. CONCLUSIONS: These results suggest that ammonia and ammonium chloride inhibit the growth of gastric cells in S phase. This mechanism may make a significant contribution to the pathogenesis of Helicobacter pylori-associated gastric mucosal atrophy.

[Higher production of tumor necrosis factor (TNF) elicited by a biological response modifier (BRM) in aging mice].

We investigated age-related changes in the capacity for tumor necrosis factor (TNF) production in young and aged inbred C3H/He mice by injecting them with OK-432, a biological response modifier (BRM). An intravenous injection of 0.4mg of OK-432 was found to induce TNF production and two consecutive injections of 2KE of OK-432 induced much higher TNF production. Both the single and two consecutive injections of OK-432 induced significantly higher TNF production in aged mice than in young ones. Furthermore, the TNF-productive response to the two consecutive injections of OK-432 seemed to increase with aging. Male mice tended to show a marginally higher TNF-productive response than females. The mechanism by which aged mice have a higher capacity for TNF production is not clear. The following possibilities are conceivable. 1) Macrophages which are major TNF producer cells may be activated in aged mice. 2) Specific T cells which are cross-reactive to antigenic determinants in OK-432 may be increased in number in aging mice and activate macrophages effectively to produce TNF when stimulated by OK-432. In general, immunological functions tend to decline with aging. Our present results, however, suggest that by using an appropriate BRM we may be able to induce higher TNF production in the aged. This might lead to effective prevention and therapy for tumors, which increase in incidence with age.

[A case control study of risk factors for Japanese cedar pollinosis].

Risk factors for Japanese cedar pollinosis including past or family history of allergic diseases, smoking and passive smoking, dwelling conditions, and life events were analyzed by a case control method. Patients with Japanese cedar pollinosis (22 males and 67 females) were matched with a corresponding number of patients without potential symptoms of pollinosis according to sex and age (+/- 5 years). The mean age was 39 years in both groups. The odds ratio (OR) was calculated by McNemar's method and the conditional logistic regression model. The design and methodology in this study were somewhat inadequate so that the validity of the results is limited. The most important problem was no-matching according to exposure to pollen. Significantly high OR for past history of allergic disease (8.80, 95% confidence interval (CI); 3.49-22.2), atopic sermatitis (9.00, 95% CI; 1.14-71.0), and a sibling history of allergic disease (3.25, 95% CI; 1.06-9.97) were consistent with former genetical studies. ORs were lower than unity for current smokers (0.36, 95% CI; 0.11-1.13) and those smoking 10 cigarettes/day or more (0.20, 95% CI; 0.04-0.91) relative to nonsmokers. The OR for passive smoking from 7-15 years of age as a result of the father's smoking habit (0.38, 95% CI; 0.17-0.86) was also significantly low. Smoking was suggested to increase the level of total and antigen-specific IgE in serum by former studies, so that sensitization and symptoms should be studied separately. The high OR of residents in a business or light industrial area (5.00, 95% CI; 1.45-17.3) suggested an association with air pollution.(ABSTRACT TRUNCATED AT 250 WORDS)

[Age-associated changes in antibody production of murine spleen cells].

In the present work, we studied age-associated changes in murine immune functions. We estimated total antibodies and autoantibodies to single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA) histone and collagen in sera and culture supernatants of spleen cells from young and aged BALB/c, C57BL/6 and MRL/MpJ-(+/+) (MRL/n) mice. In MRL/n mice, the IgM class of the total antibody level in serum increased gradually to the maximum at 3 months of age, and then started to decrease. In contrast, the IgG class started to rise with age after the age of 9 months. Serum levels of IgM and IgG autoantibodies to DNA were dominant in MRL/n mice, and the IgG class started to increase in earlier stages of life than in BALB/c and C57BL/6 mice. Anti-DNA autoantibodies were produced in lipopolysaccharide (LPS)-stimulated cultures of spleen cells from BALB/c, C57BL/6 and MRL/n mice. The stimulatory effect of LPS on autoantibody production was significantly reduced by the addition of concanavalin A (Con A) to the LPS-stimulated cultures. The Con A-induced suppressive activity increased with the donor age in MRL/n mouse spleen cells. On the other hand, total antibody production in LPS-stimulated cultures was not affected by the addition of Con A to the cultures. These results may suggest that IgG autoantibody-producing B cells increase with age in MRL/n mouse spleen cells, and that the suppressive activity on autoantibody production is selectively augmented.

[A case-control study of ulcerative colitis].

In order to evaluate the epidemiological characteristics of ulcerative colitis, we conducted a matched case-control study of ulcerative colitis. Fifty patients with ulcerative colitis diagnosed at three hospitals in Kyoto, Osaka and Hyogo Prefectures in Japan during 1984 and 1987 were interviewed about their habitual and pre-illness diets, personal habits, past histories and family histories. The results were compared to those for fifty healthy controls who were matched for sex and age (+/- 2 years) and participated in health-screening examinations in Kyoto or Osaka during the same period. Frequent intake of rice (4 or more bowls a day), bread (3 or more times a week), and green tea (7 or more cups a day) significantly increased the relative risk of ulcerative colitis. However, a past history of appendectomy was negatively correlated with the disease. There were no relationships between ulcerative colitis and consumption of animal foods and tobacco.

[Comparison of a rural town and a fishing town: diet and circulatory system diseases].

We report a comparative study of the Standardized Mortality Ratio (SMR) for circulatory system diseases and diet in a rural town and a fishing town in Kyoto Prefecture. SMR was assessed during the 5-year period from 1983 through 1987, and compared with the standard age- and sex-adjusted demographic and mortality statistics compiled by the National Census Bureau of Japan in 1985. A food frequency questionnaire in which the respondents evaluated their food consumption during the previous 1-year period was used to assess diet. The questionnaire was administered during February 1989 in the rural town and during February 1990 in the fishing town. In comparison with the standard statistics, SMR was higher in the rural town and lower in the fishing town. The inhabitants of the fishing town more frequently consumed low-fat and low-sodium foods, such as fish, potatoes, tofu, and green, yellow and other vegetables, and less frequently consumed high-fat and high-sodium foods, such as meat, fried food, pickles, than did the inhabitants of the rural town. The residents of the fishing town also consumed a greater variety of foods in one week. The two towns differ in geography and economic structure, and their inhabitants have different life-styles and eating habits. The lower SMR for circulatory system diseases in the fishing town may be related to the greater consumption of fish and vegetables with lower meat and salt intake, as well as the balanced of diet.

[A study using a food-frequency questionnaire--simultaneous discrimination of dietary patterns of anemic and hyperlipemic women].

A survey investigating the intake frequency of various foods and eating habits was made by a special form of structured questionnaire. Those who answered the questionnaire were 184 women aged from 40 to 59 living in a rural district in Kyoto Prefecture. They were divided into three blood groups according to the contents of hemoglobin (Hb) and serum cholesterol (T-CHO) as follows: Group A (Anemia): Hb less than 12 g/dl and T-CHO less than 220 mg/dl Group B (Normal): Hb greater than or equal to 12 g/dl and T-CHO less than 220 mg/dl Group C (Hyperlipidemia): Hb greater than or equal to 12 g/dl and T-CHO greater than or equal to 220 mg/dl As far as the intake frequency of each food and the eating habits of the three blood groups are concerned, there wasn't any notable difference. However, when the statistical method called "quantification theory II" was applied using the blood groups as the objective variable and the 27 items in the food frequency questionnaire, as well as the obesity index, as explanatory variables, it was found that the dietary patterns were different among the three blood groups. The result was that 68% of women in group A were discriminated correctly, as were 55% in group B, and 83% in group C. The analysis of the data from food frequency questionnaires by the "quantification theory II" statistical methods seems to be useful to reveal dietary patterns.

[A study of factors in medical insurance records associated with participation in health examinations].

Factors in medical insurance records of two groups classified as participants and nonparticipants in a multiphasic health examination (MHE) conducted in a rural town in Kyoto prefecture were compared. The purpose of this study was to clarify how the conditions of medical care influenced the participation in the MHE. The factors were days of consultation, total insurance points and days of consultation classified by specific disease and the area of the medical facility. Participants were examined at least once in 1987-1988 and nonparticipants were never examined in the corresponding period. The data were obtained from the medical insurance records of outpatients for the period from April 1986 thru March 1987. The medical care bills of 170 males and 201 females aged 30-69 were randomly sampled from National Health Insurance records (unit = family), and those of 55 males and 88 females aged 70 and over were from the Medical Service for the Aged (unit = person). These samples were about one forth of target population respectively. Both older participants and older nonparticipants of both sexes had more consultation days and more total insurance points than the corresponding younger subjects. Nonparticipants of both sexes aged 70 and over had more consultation days and more total insurance points than participants; female nonparticipants aged 50-69 had slightly more consultation days and those aged 30-49 also had more insurance points. Nonparticipants tended to have previous medical care for hypertension or ischemic heart disease, which the MHE is responsible for discovering.(ABSTRACT TRUNCATED AT 250 WORDS)