Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Linkage between dendritic and T cell commitments in human circulating hematopoietic progenitors.

The relationships between commitments of dendritic cells (DCs) and T cells in human hematopoietic stem cells are not well understood. In this study, we enumerate and characterize conventional DC and plasmacytoid DC precursors in association with T cell and thymus-derived types of NK cell precursors among CD34(+) hematopoietic progenitor cells (HPCs) circulating in human peripheral blood. By limiting-dilution analyses using coculture with stroma cells expressing Notch1 ligand, the precursor frequencies (PFs) of DCs in HPCs were found to significantly correlate with T cell PFs, but not with NK cell PFs, among healthy donors. Clonal analyses showed that the majority of T/NK dual- and T single-lineage precursors-but only a minority of NK single-lineage precursors-were associated with the generation of DC progenies. All clones producing both DC and T cell progenies were found with monocyte and/or granulocyte progenies, suggesting DC differentiation via myeloid DC pathways. Analyses of peripheral blood HPC subpopulations revealed that the lineage split between DC and T/NK cell progenitor occurs at the stage prior to bifurcation into T and NK cell lineages. The findings suggest a strong linkage between DC and T cell commitments, which may be imprinted in circulating lymphoid-primed multipotent progenitors or in more upstream HPCs.

Age-associated changes in the differentiation potentials of human circulating hematopoietic progenitors to T- or NK-lineage cells.

Age-associated changes of T and NK cell (T/NK) potential of human hematopoietic stem cells are unknown. In this study, we enumerate and characterize T/NK precursors among CD34(+)Lin(-) cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using coculture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta-like 1. The frequency of T cell precursors in CD34(+)Lin(-) cells was found to decrease with donor age, whereas the ratio of NK to T cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T to NK cell lineage with aging. Clonal analyses of CD34(+)Lin(-) cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Because nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in coculture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multipotent progenitors. The age-associated increase in NK over T cell commitment might occur in precursor cells with T/NK/myeloid potential.

Experimentally induced, synergistic late effects of a single dose of radiation and aging: significance in LKS fraction as compared with mature blood cells.

The number of murine mature blood cells recovered within 6 weeks after 2-Gy whole-body irradiation at 6 weeks of age, whereas in the case of the undifferentiated hematopoietic stem/progenitor cell (HSC/HPC) compartment [cells in the lineage-negative, c-kit-positive and stem-cell-antigen-1-positive (LKS) fraction], the numerical differences between mice with and without irradiation remained more than a year, but conclusively the cells showed numerical recovery. When mice were exposed to radiation at 6 months of age, acute damages of mature blood cells were rather milder probably because of their maturation with age; but again, cells in the LKS fraction were specifically damaged, and their numerical recovery was significantly delayed probably as a result of LKS-specific cellular damages. Interestingly, in contrast to the recovery of the number of cells in the LKS fraction, their quality was not recovered, which was quantitatively assessed on the basis of oxidative-stress-related fluorescence intensity. To investigate why the recovery in the number of cells in the LKS fraction was delayed, expression levels of genes related to cellular proliferation and apoptosis of cells in the bone marrow and LKS fraction were analyzed by real-time polymerase chain reaction (RT-PCR). In the case of 21-month-old mice after radiation exposure, Ccnd1, PiK3r1 and Fyn were overexpressed solely in cells in the LKS fraction. Because Ccnd1and PiK3r1 upregulated by aging were further upregulated by radiation, single-dose radiation seemed to induce the acceleration of aging, which is related to the essential biological responses during aging based on a lifetime-dependent relationship between a living creature and xenobiotic materials.

Evaluation of systemic markers of inflammation in atomic-bomb survivors with special reference to radiation and age effects.

Past exposure to atomic bomb (A-bomb) radiation has exerted various long-lasting deleterious effects on the health of survivors. Some of these effects are seen even after >60 yr. In this study, we evaluated the subclinical inflammatory status of 442 A-bomb survivors, in terms of 8 inflammation-related cytokines or markers, comprised of plasma levels of reactive oxygen species (ROS), interleukin (IL)-6, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), IL-4, IL-10, and immunoglobulins, and erythrocyte sedimentation rate (ESR). The effects of past radiation exposure and natural aging on these markers were individually assessed and compared. Next, to assess the biologically significant relationship between inflammation and radiation exposure or aging, which was masked by the interrelationship of those cytokines/markers, we used multivariate statistical analyses and evaluated the systemic markers of inflammation as scores being calculated by linear combinations of selected cytokines and markers. Our results indicate that a linear combination of ROS, IL-6, CRP, and ESR generated a score that was the most indicative of inflammation and revealed clear dependences on radiation dose and aging that were found to be statistically significant. The results suggest that collectively, radiation exposure, in conjunction with natural aging, may enhance the persistent inflammatory status of A-bomb survivors.

Radiation-dose response of glycophorin A somatic mutation in erythrocytes associated with gene polymorphisms of p53 binding protein 1.

Information on individual variations in response to ionizing radiation is still quite limited. Previous studies of atomic-bomb survivors revealed that somatic mutations at the glycophorin A (GPA) gene locus in erythrocytes were significantly elevated with radiation exposure dose, and that the dose response was significantly higher in survivors with subsequent cancer development compared to those without cancer development. Noteworthy in these studies were great inter-individual differences in GPA mutant fraction even in persons with similar radiation doses. It is hypothesized that persistent GPA mutations in erythrocytes of atomic-bomb survivors are derived from those in long-lived hematopoietic stem cell (HSC) populations, and that individual genetic backgrounds, specifically related to DNA double-strand break repair, contribute to individual differences in HSC mutability following radiation exposure. Thus, we examined the relationship between radiation exposure, GPA mutant fraction in erythrocytes, and single nucleotide polymorphisms (SNPs) of the key gene involved in DNA double-strand break repair, p53 binding protein 1 (53BP1). 53BP1 SNPs and inferred haplotypes demonstrated a significant interaction with radiation dose, suggesting that radiation-dose response of GPA somatic mutation is partly dependent on 53BP1 genotype. It is also possible that 53BP1 plays a significant role in DNA double-strand break repair in HSCs following radiation exposure.

Body iron stores and breast cancer risk in female atomic bomb survivors.

Iron can be a potent pro-oxidant and, on this basis, elevated body iron may increase the risk of cancer. Although epidemiological evidence is mixed, there is overall support for this possibility. In addition, because of this same oxidative capacity, body iron levels may alter radiation sensitivity. In the present study, a nested case-control study of breast cancer was conducted in Japanese atomic bomb survivors. Stored serum samples from the Adult Health Study cohort were assayed for ferritin levels and joint statistical analyses were conducted of ferritin and radiation dose on the risk of breast cancer. Serum ferritin is the best feasible indicator of body iron levels in otherwise healthy people. A total of 107 cases and 212 controls were available for analysis. The relative risk (RR) of breast cancer for a 1 log unit increase in ferritin was 1.4 (95% confidence interval 1.1-1.8). This translates to an RR of 1.64 comparing high and low values of the interquartile range among controls (58 and 13.2 ng/mL, respectively). The results support the hypothesis that elevated body iron stores increase the risk of breast cancer. However, the study was inconclusive regarding the question of whether body iron alters radiation-induced breast cancer risk.

Relationship of sFas with metabolic risk factors and their clusters.

BACKGROUND: Metabolic risk factors are known to cause atherosclerosis through inflammation. In the process of inflammation, soluble Fas (sFas) may interfere with the apoptotic pathway and contribute to dysregulated inflammation. Recent studies suggest sFas as a marker of inflammation in patients with cardiovascular diseases. However, whether a relationship exists between sFas levels and metabolic risk factors among healthy subjects remains unclear. MATERIALS AND METHODS: We measured the serum sFas levels of 876 subjects selected as controls for a nested case-control study within the JACC Study. The adjusted means of the sFas levels were compared according to the presence of overweight/obesity, hypertension, hyperlipidaemia, diabetes and their clusters. RESULTS: sFas level was significantly associated with overweight/obesity (2.42 ng mL(-1) in overweight/obese men and 2.19 in others) and hyperlipidaemia (2.34 ng mL(-1) in men with hyperlipidaemia and 2.19 in others) among men, though not with hypertension or diabetes. Moreover, a clear association between sFas levels and the cluster number of metabolic risk factors was observed independently with age, smoking and drinking(2.39, 2.28, 2.24 and 2.11 ng dL(-1) in men with three to four, two, one and none of the four metabolic risk factors respectively). However, among women, clear associations were not observed between sFas levels and the four metabolic risk factors or their clustering. CONCLUSIONS: Serum sFas levels appear to be associated with overweight/obesity, hyperlipidaemia and clusters of metabolic risk factors among men, suggesting that sFas may elevate to down-regulate increased apoptosis in atherogenesis processes.

Increased DNA damage in hematopoietic cells of mice with graft-versus-host disease.

Patients who received hematopoietic cell transplants have an increased risk for a new malignancy. In addition to genotoxic regimens such as radiotherapy and chemotherapy, graft-versus-host disease (GVHD) is a risk factor for development of new malignancies in long-term survivors. To understand mechanisms underlying this malignant transformation, we evaluated genomic damage in several murine models of GVHD by enumerating reticulocytes containing micronuclei (MN) in the blood after semi-allogeneic (parent-into-F1) hematopoietic cell transplantation. On day 40 after transplantation, MN frequencies were significantly increased in unirradiated (C57BL6 x DBA/2) F1 (BDF1) and (BALB/c x C57BL6) F1 (CBF1) mice that received cells from C57BL6 (B6) donors. MN frequencies were not significantly increased in F1 mice that received cells from DBA/2 or BALB/c donors. Serum levels of tumor necrosis factor-alpha (TNF-alpha) were higher after transplantation with B6 donors than with DBA/2 or BALB/c donors. The results indicate that GVHD, without irradiation, can induce genomic damage associated with inflammatory reactions manifested by increased TNF-alpha levels.

Lung cancer susceptibility among atomic bomb survivors in relation to CA repeat number polymorphism of epidermal growth factor receptor gene and radiation dose.

Lung cancer is a leading cause of cancer death worldwide. Prevention could be improved by identifying susceptible individuals as well as improving understanding of interactions between genes and etiological environmental agents, including radiation exposure. The epidermal growth factor receptor (EGFR)-signaling pathway, regulating cellular radiation sensitivity, is an oncogenic cascade involved in lung cancer, especially adenocarcinoma. The cytosine adenine (CA) repeat number polymorphism in the first intron of EGFR has been shown to be inversely correlated with EGFR production. It is hypothesized that CA repeat number may modulate individual susceptibility to lung cancer. Thus, we carried out a case-cohort study within the Japanese atomic bomb (A-bomb) survivor cohort to evaluate a possible association of CA repeat polymorphism with lung cancer risk in radiation-exposed or negligibly exposed (<5 mGy) A-bomb survivors. First, by dividing study subjects into Short and Long genotypes, defined as the summed CA repeat number of two alleles < or = 37 and > or = 38, respectively, we found that the Short genotype was significantly associated with an increased risk of lung cancer, specifically adenocarcinoma, among negligibly exposed subjects. Next, we found that prior radiation exposure significantly enhanced lung cancer risk of survivors with the Long genotype, whereas the risk for the Short genotype did not show any significant increase with radiation dose, resulting in indistinguishable risks between these genotypes at a high radiation dose. Our findings imply that the EGFR pathway plays a crucial role in assessing individual susceptibility to lung adenocarcinoma in relation to radiation exposure.

Characteristic gene expression in stromal cells of gastric cancers among atomic-bomb survivors.

To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic-bomb survivors is important. In our study, we developed a custom oligonucleotide array of 208 genes. We analyzed gene expression profiles of gastric cancers (GCs) from atomic-bomb survivors and identified 9 genes with significantly lower expression in GCs from exposed patients than in GCs from nonexposed patients. Among these 9 genes, expression of versican and osteonectin was investigated in greater detail using immunohistochemistry in 116 GCs from 64 exposed and 52 nonexposed patients who developed GC after the bombing. In the Stage I/II GCs, the clinicopathologic, phenotypic and proliferative characteristics of GCs from exposed and nonexposed patients did not differ significantly; however, versican and osteonectin were expressed at much lower levels in the area of tumor-associated stroma of exposed patients than in nonexposed patients (p = 0.026 and p = 0.024, respectively). These results suggest that the characteristics of tumor-associated stromal cells differ between GCs from exposed and nonexposed patients.

DNA methylation status is inversely correlated with green tea intake and physical activity in gastric cancer patients.

Epigenetic silencing of genes by aberrant DNA methylation is recognized as a crucial component of the mechanism underlying tumorigenesis. However, the relationship between DNA methylation and the past lifestyle in cancer patients remains largely unknown. We examined the methylation statuses of 6 tumor-related genes, CDX2 (homeobox transcription factor), BMP-2 (bone morphogenetic protein 2), p16 (INK4A), CACNA2D3 (calcium channel-related), GATA-5 (transcription factor) and ER (estrogen receptor), in 106 primary gastric carcinomas by methylation-specific PCR and compared them with the past lifestyles of the patients. The methylation frequencies of the genes were 23.6, 21.7, 9.4, 32.4, 40.8 and 59.1%, respectively. Significant association was found between a decreased intake of green tea and methylation of CDX2 and BMP-2. More physical activity was correlated with a lower methylation frequency of CACNA2D3. Of these 6 genes, the methylation statuses of CDX2, BMP-2 and p16 revealed a significant interrelationship and those of CACNA2D3, GATA-5 and ER did likewise. Thus, some epidemiological factors, such as green tea intake, could be important as to determination of the methylation statuses of selected genes and may influence the development of cancer, including that of the stomach.

Caspase-independent cell death without generation of reactive oxygen species in irradiated MOLT-4 human leukemia cells.

To improve our understanding of ionizing radiation effects on immune cells, we investigated steps leading to radiation-induced cell death in MOLT-4, a thymus-derived human leukemia cell. After exposure of MOLT-4 cells to 4 Gy of X-rays, irradiated cells sequentially showed increase in intracellular reactive oxygen species (ROS), decrease in mitochondrial membrane potential, and eventually apoptotic cell death. In the presence of the caspase inhibitor z-VAD-fmk, irradiated cells exhibited necrotic characteristics such as mitochondrial swelling instead of apoptosis. ROS generation was not detected during this necrotic cell death process. These results indicate that radiation-induced apoptosis in MOLT-4 cells requires elevation of intracellular ROS as well as activation of a series of caspases, whereas the cryptic necrosis program--which is independent of intracellular ROS generation and caspase activation--is activated when the apoptosis pathway is blocked.

Cigarette smoking and serum soluble Fas levels: Findings from the JACC study.

Cigarette smoking enhances low-grade systemic inflammation in the lung and other organs. Activated immune cells play an important role at early and late stages of inflammation, and in recent years, soluble Fas (sFas), an isoform of death molecule Fas, was found to interfere with the apoptotic pathways of these activated immune cells. The aim of this study was to confirm the association between cigarette smoking and sFas levels in healthy male subjects. We measured serum sFas levels of 4415 male subjects selected as controls for a nested case-control study within the large-scale cohort study conducted in Japan, called the JACC Study. Smoking status at baseline was evaluated by a self-administered questionnaire. Least square means of sFas according to smoking status and numbers of cigarettes smoked per day among smokers were calculated and adjusted for possible confounding factors. Mean sFas levels showed an increasing trend across never smokers, past smokers and current smokers, as 2.21 (95% CI: 2.14-2.27) ng/ml, 2.29 (2.22-2.36) ng/ml, and 2.36 (2.30-2.43) ng/ml, respectively. However, no dose-response relationship was observed between the number of cigarettes smoked per day and sFas levels among smokers.

Cytolethal distending toxin induces caspase-dependent and -independent cell death in MOLT-4 cells.

Cytolethal distending toxin (CDT) induces apoptosis using the caspase-dependent classical pathway in the majority of human leukemic T cells (MOLT-4). However, we found the process to cell death is only partially inhibited by pretreatment of the cells with a general caspase inhibitor, z-VAD-fmk. Flow cytometric analysis using annexin V and propidium iodide showed that a 48-h CDT treatment decreased the living cell population by 35% even in the presence of z-VAD-fmk. z-VAD-fmk completely inhibited caspase activity in 24 h CDT-intoxicated cells. Further, CDT with z-VAD-fmk treatment clearly increased the cell population that had a low level of intracellular reactive oxygen. This is a characteristic opposite to that of caspase-dependent apoptosis. Overexpression of bcl2 almost completely inhibited cell death using CDT treatment in the presence of z-VAD-fmk. The data suggest there are at least two different pathways used in CDT-induced cell death: conventional caspase-dependent (early) apoptotic cell death and caspase-independent (late) death. Both occur via the mitochondrial membrane disruption pathway.

Soluble Fas level and cancer mortality: findings from a nested case-control study within a large-scale prospective study.

Soluble Fas (sFas) is known to play an important role in the development of cancers of various sites. To confirm whether or not the serum sFas level can be a predictor of cancer, we conducted a nested case-control study within a large-scale population-based cohort study in Japan. Serum samples were collected from 39,242 participants (13,839 men and 25,403 women) at baseline, all of whom were followed until 1997 for mortality and until 1994 for cancer incidence. Three controls were randomly selected and matched to each cancer case for gender, age and residential area. Serum values of sFas were measured by enzyme-linked immuno-adsorbent assay, using commercially available kits. The odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated using conditional logistic models, based on 798 total cancer mortality cases and their 2,353 matched controls. The risk of total cancer mortality was increased according to sFas levels, and the OR of the highest quartile compared with that of the lowest was 1.81 (95% CI; 1.40-2.34) after adjusting for smoking and drinking status, and body mass index. This positive association remained unaltered when cases were divided into 2 groups according to the observation period. Our results suggest that serum sFas has a possibility to detect people at high risk for cancer prior to diagnosis, since it increased before cancer diagnosis in those apparently healthy people.

Exon 19 of EGFR mutation in relation to the CA-repeat polymorphism in intron 1.

Epidermal growth factor receptor (EGFR) mutations in lung cancer enhance tyrosine kinase activity and increase sensitivity to the EGFR tyrosine kinase inhibitor, gefitinib. Mutation analysis of the EGFR gene is therefore indispensable for predicting gefitinib response. We investigated a CA-repeat polymorphism in the EGFR gene related to EGFR mutations. Because an increasing number of CA-repeats at intron 1 of the EGFR gene has been reported to reduce transcription activity, we examined the relationship between EGFR mutations and this CA-repeat polymorphism. EGFR mutations at exon 19 were closely associated with shorter CA-repeat length in the shorter allele, but this was not the case for EGFR mutations at exons 18 or 21. Increased intrinsic EGFR mRNA expression in non-cancerous lung tissues from lung adenocarcinoma patients was also significantly associated with shorter CA-repeat length. A higher frequency of EGFR mutations at exon 19 was associated with shorter CA-repeat length only in patients with high levels of EGFR mRNA expression. To determine the phenotypes of cells possessing shorter CA-repeats, an in vitro study using human bronchial epithelial cells with different CA-repeat lengths was performed; more rapid cell growth and activated EGF/EGFR signaling were found more often in the cells having both shorter CA-repeats and increased EGFR mRNA expression. These results suggest that CA-repeat length in the EGFR gene may be a genetic factor related to cancer in the case of EGFR mutations at exon 19. The mechanism likely involves enhanced intrinsic expression of EGFR mRNA and activated EGF/EGFR signaling that accompany shorter CA-repeats.

Sixty years of follow-up of Hiroshima and Nagasaki survivors: current progress in molecular epidemiology studies.

This article provides an overview of the on-going molecular epidemiology studies among atomic-bomb survivors conducted at the Radiation Effects Research Foundation in Japan. The focus is on: (a) inter-individual variations in sensitivity to radiation-induced somatic mutations (glycophorin A (GPA) mutations) and their potential relevance to differences in susceptibility to radiation-related cancers and (b) the role of specific mutations/rearrangements in radiation-induced thyroid and colorectal cancers. The glycophorin A mutant fractions showed large differences between the survivors at each of the estimated bone marrow doses. Of note is the finding at doses>or=1 Gy; that the slope of the mutant fraction was significantly higher in the 'cancer group' than in the 'non-cancer group'. This study provided the basis for validating the use of gammaH2AX and reticulocyte micronucleus assays for evaluating radiosensitivity differences and genetic instability, respectively, in our studies in the coming years. Preliminary results from our molecular oncology studies on adult-onset papillary thyroid cancer provide evidence for the induction of RET/PTC rearrangements and BRAF point mutation (both known to be early stage events in adult-onset papillary thyroid cancer) but with a difference: cases associated with the rearrangements were more frequent at high doses, and developed sooner than those with BRAF mutation. In the case of colorectal cancer, the results suggest that radiation exposure might influence microsatellite instability (MSI) status through MSI-related epigenetic and genetic alterations-processes that might occur in the early stage of colorectal carcinogenesis.

Adaptation to estradiol deprivation causes up-regulation of growth factor pathways and hypersensitivity to estradiol in breast cancer cells.

Deprivation of estrogen causes breast tumors in women to adapt and develop enhanced sensitivity to this steroid. Accordingly, women relapsing after treatment with oophorectomy, which substantially lowers estradiol for a prolonged period, respond secondarily to aromatase inhibitors with tumor regression. We have utilized in vitro and in vivo model systems to examine the biologic processes whereby Long Term Estradiol Deprivation (LTED) causes cells to adapt and develop hypersensitivity to estradiol. Several mechanisms are associated with this response including up-regulation of ERalpha and the MAP kinase, PI-3-kinase and mTOR growth factor pathways. ERalpha is 4-10 fold up-regulated as a result of demethylation of its C promoter, This nuclear receptor then co-opts a classical growth factor pathway using SHC, Grb-2 and Sos. This induces rapid nongenomic effects which are enhanced in LTED cells. The molecules involved in the nongenomic signaling process have been identified. Estradiol binds to cell membrane-associated ERalpha which physically associates with the adaptor protein SHC and induces its phosphorylation. In turn, SHC binds Grb-2 and Sos which results in the rapid activation of MAP kinase. These nongenomic effects ofestradiol produce biologic effects as evidenced by Elk-1 activation and by morphologic changes in cell membranes. Additional effects include activation of the PI-3-kinase and mTOR pathways through estradiol-induced binding of ERalpha to the IGF-1 and EGF receptors. A major question is how ERalpha locates in the plasma membrane since it does not contain an inherent membrane localization signal. We have provided evidence that the IGF-1 receptor serves as an anchor for ERalpha in the plasma membrane. Estradiol causes phosphorylation of the adaptor protein, SHC and the IGF-1 receptor itself. SHC, after binding to ERalpha, serves as the "glue" which tethers ERalpha to SHC binding sites on the activated IFG-1 receptors. Use of siRNA methodology to knock down SHC allows the conclusion that SHC is needed for ERalpha to localize in the plasma membrane. In order to abrogate growth factor induced hypersensitivity, we have utilized a drug, farnesylthiosalicylic acid, which blocks the binding of GTP-Ras to its membrane acceptor protein, galectin 1 and reduces the activation of MAP kinase. We have shown that this drug is a potent inhibitor of mTOR and this provides the major means for inhibition of cell proliferation. The concept of "adaptive hypersensitivity" and the mechanisms responsible for this phenomenon have important clinical implications. The efficacy ofaromatase inhibitors in patients relapsing on tamoxifen could be explained by this mechanism and inhibitors of growth factor pathways should reverse the hypersensitivity phenomenon and result in prolongation of the efficacy of hormonal therapy for breast cancer.

Protective effect of vitamin C on oxidative stress: a randomized controlled trial.

BACKGROUND: Although a number of reports regarding the role of reactive oxygen species (ROS) as the first step in cancer induction exist, few studies have investigated how vitamin C influences ROS in human plasma. AIM OF THE STUDY: Using the ROS assay system, a method recently established by one of the authors, we aimed to evaluate the effect of vitamin C supplementation on serum ROS among subjects diagnosed with chronic gastritis. METHODS: A total of 244 Japanese subjects with atrophic gastritis were randomized to take 5-year supplementation of either 50 mg or 500 mg of vitamin C. RESULTS: The adjusted difference in the changes of total ROS between baseline and after 5-year supplementation was statistically significant between the intervention groups: 2.70 decrease (corresponds to 1.26% decrease) in the high-dose group and 4.16 increase (corresponds to 3.79% increase) in the low-dose group, p for difference = 0.01. CONCLUSION: Vitamin C was suggested to reduce oxidative stress among subjects with atrophic gastritis.