Effects of subchronic inhalation exposure to ethyl tertiary butyl ether on splenocytes in mice.

Ethyl tertiary-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. The current use of ETBE in gasoline or petrol is modest but increasing. To investigate the effects of ETBE on splenocytes, mice were exposed to 0 (control), 500 ppm, 1750 ppm, or 5000 ppm of ETBE by inhalation for 6 h/day for 5 days/wk over a 6- or 13-week period. Splenocytes were harvested from the control and exposed mice, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PerCP-Cy5.5-CD45R/B220), (2) T cells (PerCP-Cy5-CD3e), (3) T cell subsets (FITC-CD4 and PE-CD8a), (4) natural killer (NK) cells (PE-NK1.1), and (5) macrophages (FITC-CD11b). Body weight and the weight of the spleen were also examined. ETBE-exposure did not affect the weight of the spleen or body weight, while it transiently increased the number of RBC and the Hb concentration. The numbers of splenic CD3+, CD4+, and CD8+ T cells, the percentage of CD4+ T cells and the CD4+/CD8+ T cell ratio in the ETBE-exposed groups were significantly decreased in a dose-dependent manner. However, ETBE exposure did not affect the numbers of splenic NK cells, B cells, or macrophages or the total number of splenocytes. The above findings indicate that ETBE selectively affects the number of splenic T cells in mice.

The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements.

The constitutional t(11;22)(q23;q11) is the most common recurrent non-Robertsonian translocation in humans. The breakpoint sequences of both chromosomes are characterized by several hundred base pairs of palindromic AT-rich repeats (PATRRs). Similar PATRRs have also been identified at the breakpoints of other nonrecurrent translocations, suggesting that PATRR-mediated chromosomal translocation represents one of the universal pathways for gross chromosomal rearrangement in the human genome. We propose that PATRRs have the potential to form cruciform structures through intrastrand-base pairing in single-stranded DNA, creating a source of genomic instability and leading to translocations. Indeed, de novo examples of the t(11;22) are detected at a high frequency in sperm from normal healthy males. This review synthesizes recent data illustrating a novel paradigm for an apparent spermatogenesis-specific translocation mechanism. This observation has important implications pertaining to the predominantly paternal origin of de novo gross chromosomal rearrangements in humans.

Analysis of VH gene rearrangement and somatic hypermutation in Sjogren's syndrome and IgG4-related sclerosing sialadenitis.

IgG4-related sclerosing sialadenitis is currently considered as an autoimmune disease distinct from Sjogren's syndrome (SS) and responds extremely well to steroid therapy. To further elucidate the characteristics of IgG4-related sclerosing sialadenitis, we analysed VH fragments of IgH genes and their somatic hypermutation in SS (n = 3) and IgG4-related sclerosing sialadenitis (n = 3), using sialolithiasis (n = 3) as a non-autoimmune control. DNA was extracted from the affected inflammatory lesions. After PCR amplification of rearranged IgH genes, at least 50 clones per case (more than 500 clones in total) were sequenced for VH fragments. Monoclonal IgH rearrangement was not detected in any cases examined. When compared with sialolithiasis, there was no VH family or VH fragment specific to SS or IgG4-related sclerosing sialadenitis. However, rates of unmutated VH fragments in SS (30%) and IgG4-related sclerosing sialadenitis (39%) were higher than that in sialolithiasis (14%) with statistical significance (P = 0.0005 and P < 0.0001, respectively). This finding suggests that some autoantibodies encoded by germline or less mutated VH genes may fail to be eliminated and could play a role in the development of SS and IgG4-related sclerosing sialadenitis.

A day trip to a forest park increases human natural killer activity and the expression of anti-cancer proteins in male subjects.

We previously reported that 2-night/3-day trips to forest parks enhanced human NK activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that this increased NK activity lasted for more than 7 days after the trip in both male and female subjects. In the present study, we investigated the effect of a day trip to a forest park on human NK activity in male subjects. Twelve healthy male subjects, aged 35-53 years, were selected after giving informed consent. The subjects experienced a day trip to a forest park in the suburbs of Tokyo. They walked for two hours in the morning and afternoon, respectively, in the forest park on Sunday. Blood and urine were sampled in the morning of the following day and 7 days after the trip, and the NK activity, numbers of NK and T cells, and granulysin, perforin, and granzyme A/B-expressing lymphocytes, the concentration of cortisol in blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trip on a weekend day as the control. Phytoncide concentrations in the forest were measured. The day trip to the forest park significantly increased NK activity and the numbers of CD16(+) and CD56(+) NK cells, perforin, granulysin, and granzyme A/B-expressing NK cells and significantly decreased CD4(+) T cells, the concentrations of cortisol in the blood and adrenaline in urine. The increased NK activity lasted for 7 days after the trip. Phytoncides, such as isoprene, alpha-pinene, and beta-pinene, were detected in the forest air. These findings indicate that the day trip to the forest park also increased the NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted for at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone levels may partially contribute to the increased NK activity.

Association between Oral Frailty and Nutritional Status among Community-Dwelling Older Adults: the Takashimadaira Study.

OBJECTIVES: Recently, the concept of oral frailty, defined as accumulated deficits in oral health, has been introduced in Japan. However, data about its association with nutritional status are limited. Thus, this cross-sectional study aimed to investigate the association between oral frailty and malnutrition among community-dwelling older adults. DESIGN: Cross-sectional study. SETTING: Community. PARTICIPANTS: One thousand and fifty-four individuals (428 men and 626 women, mean age: 77.0 years) from the Takashimadaira Study. MEASUREMENTS: Based on a multifaceted oral health assessment, oral frailty was defined as greater than or equal to three of the following components: (1) low number of remaining teeth, (2) decreased masticatory performance, (3) reduced articulatory oral motor skill, (4) low tongue pressure, and difficulties in (5) eating and (6) swallowing. The nutritional status was evaluated using the Mini Nutritional Assessment®-Short Form (MNA®-SF) and serum albumin. An ordinal logistic regression model was used to evaluate the association between oral frailty and nutritional status. RESULTS: Oral frailty was observed in 217 (20.4%) participants. After adjusting for potential confounders, the participants with oral frailty had higher odds of more severe malnutrition evaluated using MNA®-SF (adjusted odds ratio: 2.17; 95% confidence interval: 1.58-2.98) and serum albumin level (adjusted odds ratio: 1.59; 95% confidence interval: 1.10-2.31). CONCLUSION: Oral frailty was associated with nutritional status among Japanese older adults. Maintaining comprehensive oral health and function may be effective for malnutrition prevention in community-dwelling older adults. However, further studies must be conducted to validate the generalizability of the results of the current study.

Association of Dietary Variety and Appetite with Sleep Quality in Urban-Dwelling Older Japanese Adults.

OBJECTIVES: Although it has been shown that specific foods and nutrients are associated with sleep quality, few studies have examined the association of dietary variety and appetite with sleep quality in older adults. DESIGN AND SETTING: A cross-sectional study was conducted that examined the association of dietary variety and appetite with sleep quality in Japanese adults aged ≥70 years who resided in the metropolitan area of Tokyo, Japan. PARTICIPANTS: Data were collected in two steps: a mailed interview survey and an on-site survey. Those who responded to the surveys and met the inclusion criteria were included. MEASUREMENTS: Dietary variety, appetite, and sleep quality were assessed using a Dietary Variety Score (DVS), Council on Nutrition Appetite Questionnaire (CNAQ) score, and sleep efficiency, respectively. The sleep efficiency is the ratio of sleep duration to total time in bed (retiring time-awakening time). We defined the individuals with a sleep efficiency less than 75% as having poor sleep quality. RESULTS: Mean DVS and CNAQ score were 3.8 and 29.6 points, respectively. The rate of individuals with poor sleep quality was 11.7%. In the fully adjusted model, the odds ratios (OR) for low sleep efficiency in the middle and highest group categories of the DVS were 0.83 (95% confidence interval [CI], 0.54-1.29) and 0.50 (95% CI, 0.28-0.90), respectively, in reference to the lowest group category (p for trend = 0.023). The OR for low sleep efficiency in the middle and highest group categories of the CNAQ score were 0.73 (95% CI, 0.47-1.14) and 0.54 (95% CI, 0.30-0.96), respectively, in reference to the lowest group category (p for trend = 0.031). CONCLUSIONS: The higher DVS and CNAQ scores were significantly associated with higher sleep efficiency. Thus, dietary variety and good appetite might help maintain good sleep quality in urban-dwelling older Japanese adults.

Effect of phytoncide from trees on human natural killer cell function.

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37-60 years, who stayed at an urban hotel for 3 nights from 7.00 p.m. to 8.00 a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

The relationship between n-3 long-chain polyunsaturated fatty acids and pulse wave velocity in diabetic and non-diabetic patients under long-term hemodialysis. A horizontal study.

BACKGROUND: Diabetes mellitus (DM) and deficiency in n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) are known to increase the incidence of cardiovascular disease (CVD). However, it has not yet been reported whether n-3 LCPUFAs are related to arteriosclerosis in patients under long-term hemodialysis (HD). METHODS: Pulse wave velocity from the brachium to the ankle (baPWV) was measured as a marker of arteriosclerosis with a volume-plethysmographic apparatus in 147 long-term HD patients (non-diabetic (non-DM): 51 males/42 females, 62 +/- 14 y; and DM: 33 males/21 females, 67 +/- 9 y). The fatty acid composition of the total phospholipid fraction from washed RBCs was analyzed by gas chromatography. Analyses were adjusted for age, sex, diastolic blood pressure, pulse, body mass index, duration of HD treatment, smoking status, LDL/HDL-cholesterol ratios and diabetes mellitus (DM). RESULTS: The mean baPWV was 18.9 +/- 5.2 and 23.7 +/- 6.3 m/s in non-DM and DM patients, respectively. The mean baPWV in DM patients was significantly higher than that of non-DM patients after adjustment (p = 0.0002). Multiple regression analysis showed that there was a significant inverse association between baPWV and docosahexaenoic acid (DHA) levels (p = 0.017) and DHA/arachidonic acid (AA) ratios (p = 0.012) in RBC in non-DM patients after adjustment but not in DM patients. CONCLUSIONS: We suggest that n-3 LCPUFAs may be a negative risk factor of CVD also in non-DM HD patients. In DM patients the effects of n-3 PUFAs on the vascular system became undetectable probably because DM overwhelmingly affected PWV. Further studies in a prospective manner are necessary.

Oral gavage of capsaicin causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase of locomotor activity in the mouse.

Capsaicin (CAP), the pungent ingredient of hot red pepper, is a selective ligand for the heat-sensitive transient receptor potential V1 cation channel 1 (TRPV1). Although CAP has been traditionally used as the ingredient of spices for various foods in the world, the effect of oral intake of CAP on thermoregulation and locomotor activity, and CAP-induced activation of brain neural circuits are not well understood. In this study, therefore, we examined the effects of oral gavage of CAP on core body and tail surface temperature, locomotor activity, and Fos expression in thermoregulation- and sensory information-associated hypothalamic and medullary brain regions using freely moving mice. Oral gavage of CAP acutely decreased core body temperature and alternatively increased tail surface temperature of wild type (WT) mice, whereas such acute temperature changes were not observed in TRPV1 knockout (KO) animals. Moreover, a long-lasting increase of locomotor activity was observed in both WT and TRPV1 KO mice after oral gavage of CAP, but increase in core body temperature was seen only in TRPV1 KO animals. Oral gavage of CAP induced neuronal Fos expression in the circumventricular organs, median and medial preoptic area, arcuate nucleus, and nucleus of the solitary tract, whereas neuronal Fos expression was scarcely observed in TRPV1 KO mice. Thus, the present study demonstrates in the mice that oral intake of CAP causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase of locomotor activity, and moreover activates the brain circuits controlling thermoregulation and metabolism.

Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins.

We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing trip increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone may partially contribute to the increased NK activity.

A forest bathing trip increases human natural killer activity and expression of anti-cancer proteins in female subjects.

We previously reported that forest bathing trips enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after the trip in male subjects. In the present study, we investigated the effect of forest bathing trip on human NK activity in female subjects. Thirteen healthy nurses, age 25-43 years, professional career 4-18 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields. On day 1, the subjects walked for two hours in the afternoon in a forest field; on day 2, they walked for two hours each in the morning and afternoon in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood and completing a questionnaire. Blood and urine were sampled on the second and third days during the trip, and on days 7 and 30 after the trip. NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, the concentrations of estradiol and progesterone in serum, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the trip on a normal working day. The concentrations of phytoncides in the forests were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air. These findings indicate that a forest bathing trip also increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins in female subjects, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone levels may partially contribute to the increased NK activity.

Forest bathing enhances human natural killer activity and expression of anti-cancer proteins.

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

The CCR4 as a novel-specific molecular target for immunotherapy in Hodgkin lymphoma.

Here, we report that tumor cells from some patients (23.8%) with Hodgkin lymphoma (HL) are positive for CC chemokine receptor 4 (CCR4). We therefore tested the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to enhance antibody-dependent cellular cytotoxicity (ADCC), as a novel immunotherapy for refractory HL. KM2760 demonstrated a promising antitumor activity in the CCR4-positive HL-bearing mouse model in the therapeutic setting. Although KM2760 did not induce any ADCC mediated by mouse natural killer (NK) cells, it significantly enhanced phagocytosis mediated by mouse monocytes/macrophages against the CCR4-positive HL cell line in vitro. Together with the findings that KM2760 did not exhibit any complement-dependent cytotoxicity or direct antiproliferation activity in vitro, these data indicated that KM2760 exerted its robust in vivo antitumor activity via monocytes/macrophages in mice. In the human system, KM2760 enhanced phagocytic activity mediated by monocytes/macrophages. Furthermore, it induced robust ADCC mediated by NK cells against the CCR4-positive HL cell line in vitro. Thus, it is conceivable that KM2760 would have much more potent antitumor activity in humans than in mice. Collectively, this study strongly indicates that anti-CCR4 mAb could be a novel treatment modality for patients with CCR4-positive HL.

Photon emissions from rice cells elicited by N-acetylchitooligosaccharide are generated through phospholipid signaling in close association with the production of reactive oxygen species.

Biophotons are ultraweak light emissions from biochemical reactions in a living body. They increase in suspension-cultured rice (Oryza sativa L.) cells when elicited by N-acetylchitooligosaccharide. Biochemical analyses were undertaken to investigate the relationship between disease response and biophotons in order to clarify the emission mechanism of biophotons caused by this elicitor. Photon emissions induced by N-acetylchitohexaose were suppressed when cells were pretreated with the reactive oxygen species (ROS)-generating inhibitors: pyrocatechol-3,5-disulfonic acid disodium salt (Tiron); diphenylene iodonium (DPI); and salicylhydroxamic acid (SHAM). Conversely, exogenously applied ROS (superoxide and hydrogen peroxide) were able to induce photon emissions. The effects of protein phosphorylation (K-252a) and the Ca(2+) signaling inhibitors, ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and LaCl(3), caused photon emissions to decrease. It is clear that photon emissions from rice cells elicited by N-acetylchitohexaose are closely associated with the ROS-generating system, and are regulated by Ca(2+) signaling and protein phosphorylation. Exogenously applied phosphatidic acid (PA), the second messenger in the signal transduction of disease response, raised photon emissions in rice cells. Comparisons of photon emissions from PA and N-acetylchitohexaose regarding time courses, spectral compositions, and the inhibition ratios of several inhibitors, as well as a loss- and gain-of-function assay using the protein synthesis inhibitor cycloheximide (CHX) and PA, showed the possibility that photon emissions from rice cells elicited by N-acetylchitooligosaccharide were generated through PA, an intermediate of phospholipid signaling.

Multiple myeloma oncogene 1 (MUM1)/interferon regulatory factor 4 (IRF4) upregulates monokine induced by interferon-gamma (MIG) gene expression in B-cell malignancy.

MUM1 (multiple myeloma oncogene 1)/IRF4 (interferon regulatory factor 4) is a transcription factor that is activated as a result of t(6;14)(p25;q32) in multiple myeloma. MUM1 expression is seen in various B-cell lymphomas and predicts an unfavorable outcome in some lymphoma subtypes. To elucidate its role in B-cell malignancies, we prepared MUM1-expressing Ba/F3 cells, which proliferated until higher cellular density than the parental cells, and performed cDNA microarray analysis to identify genes whose expression is regulated by MUM1. We found that the expression of four genes including FK506-binding protein 3 (FKBP3), the monokine induced by interferon-gamma(MIG), Fas apoptotic inhibitory molecule (Faim) and Zinc-finger protein 94 was altered in the MUM1-expressing cells. We then focused on MIG since its expression was immediately upregulated by MUM1. In reporter assays, MUM1 activated the MIG promoter in cooperation with PU.1, and the interaction between MUM1 and the MIG promoter sequence was confirmed. The expression of MIG was correlated with that of MUM1 in B-CLL cell lines, and treatment with neutralizing antibodies against MIG and its receptor, CXCR3, slightly inhibited the proliferation of two MUM1-expressing lines. These results suggest that MUM1 plays roles in the progression of B-cell lymphoma/leukemia by regulating the expression of various genes including MIG. Leukemia (2005) 19, 1471-1478. doi:10.1038/sj.leu.2403833; published online 16 June 2005.

Do personality characteristics predict longevity? Findings from the Tokyo Centenarian Study.

To explore whether personality influences longevity we examined the personality characteristics of centenarians. We developed a new method that compares an actual personality test score for centenarians with a predicted test score for a 100-year-old, calculated from younger controls. The participants consisted of 70 cognitively intact Japanese centenarians aged 100-106 years and 1812 elderly people aged 60-84 years, all residents of Tokyo. The NEO five factor inventory (NEO-FFI) was used to assess the "big five" personality traits: neuroticism, extraversion, openness, agreeableness, and conscientiousness. The results showed higher openness in both male and female centenarians, and higher conscientiousness and extraversion in female centenarians, as compared to controls. These results suggest that high scores in the specific personality traits conscientiousness, extraversion, and openness, are associated with longevity. We speculate that these personality traits contribute to longevity through health-related behavior, stress reduction, and adaptation to the challenging problems of the "oldest old".

Growth-inhibitory action of an estrogen-chlorambucil conjugate (KM2210) in human breast cancer cell line MCF-7: its relation to reduction of estrogen receptor and transforming growth factor-alpha secretion.

We investigated the effects of a benzoate of an estradiol-chlorambucil conjugate (KM2210) and chlorambucil on growth, estrogen receptor, and secretion of transforming growth factor (TGF)-alpha in the hormone-dependent human breast cancer cell line MCF-7. In the presence of 10(-10)-10(-6) M KM2210, the estrogen-induced growth of MCF-7 was completely inhibited. Inhibited growth of MCF-7 treated with 10(-8) or 10(-6) M KM2210 for 4 days was not rescued by removal of the drug and the addition of estradiol. By treatment of MCF-7 with KM2210 for 4 days, estrogen receptor-binding sites were decreased at 10(-8) M and were not detected at 10(-6) M but were unaltered by 10(-8) M chlorambucil. Moreover, estrogen receptor immunoreactivity and the level of estrogen receptor mRNA were decreased through treatment with 10(-6) M KM2210 for 4 days. These suppressions occurred prior to the onset of inhibitory action on MCF-7 growth. Secretion of TGF-alpha from MCF-7 was decreased by 4 days of treatment with 10(-8) and 10(-6) M KM2210 but not with chlorambucil. The addition of exogenous TGF-alpha generally restored the growth of MCF-7 treated with 10(-8) M KM2210. We concluded that KM2210 has irreversible or at least long-standing inhibitory effect on estrogen-dependent growth of MCF-7. It is conceivable that the decrease of estrogen receptor renders the cell unable to respond to estrogen with increased TGF-alpha secretion and succeeding cell growth.

Inhibition of estradiol uptake and transforming growth factor alpha secretion in human breast cancer cell line MCF-7 by an alkyl-lysophospholipid.

We investigated the effect of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl-lysophospholipid, on the uptake of estrogen, the secretion of transforming growth factor (TGF) alpha and the content of progesterone receptors (PRs) in the hormone-dependent breast cancer cell line, MCF-7. The uptake of labeled estradiol by MCF-7 was dose dependently decreased by 12 h pretreatment with 10-25 micrograms/ml ET-18-OCH3, and this suppression occurred prior to the onset of the inhibitory action of ET-18-OCH3 on MCF-7 growth. Scatchard analysis demonstrated that ET-18-OCH3 reduced the number of estrogen receptors in MCF-7 without affecting their affinity. Both the secretion of TGF-alpha from MCF-7 into the conditioned medium and the PR content of MCF-7 were decreased by 48 h treatment with 10 micrograms/ml ET-18-OCH3. The estradiol uptake, the TGF-alpha secretion, and the PR content were not affected by platelet-activating factor, lyso-PAF, and palmitoyl-lysophosphatidylcholine, all at 10 micrograms/ml. These results suggest that the reduction of estrogen receptor level induced by ET-18-OCH3 resulted in decreases in both the secretion of TGF-alpha and the content of PR in MCF-7, and these effects are specific to ET-18-OCH3. We concluded that these effects of ET-18-OCH3 may lead, at least partly, to its antitumor action in hormone-dependent breast cancer cell lines.