Probiotic Escherichia coli Nissle 1917 alleviates the neurotoxicity caused by acrylamide in zebrafish.

Neurotoxicity is caused by damage to the brain tissue by neurotoxic agents present in the environment and artificial substances produced by human beings. Acrylamide (ACR) is one such chemical substance that causes neurotoxicity, affecting the brain cells. This neurotoxicity causes damage to the sensory and metabolic functions. The current research investigates the favourable effect of probiotic EcN (Escherichia coli Nissle 1917) on ACR-induced neurotoxicity in zebrafish. The protective role of EcN against ACR induced toxicity was assessed based on behaviour, biochemical, and gene expression analysis. Initially, the colonisation period of EcN in the zebrafish gut was determined and EcN was given orally to the zebrafish only once prior to the ACR treatment. Very interestingly, this dosage was able to ameliorate the adverse effects of ACR significantly in the brain cells. Quantification of oxidative stress and neuronal cell death clearly vindicate the efficiency of probiotic EcN in reversing the damages caused by ACR. EcN is being explored largely in recent days for its therapeutic applications. This study strongly supports the view that EcN can be developed as a supplement to the patients diagnosed with neuronal cell toxicity.

The mutant p53 mouse as a pre-clinical model.

The p53 tumor-suppressor pathway is dismantled in the development of most cancers. Mice with various p53 mutant alleles either singly or in combination with other genetic alterations are predisposed to tumor development. Here, we review studies utilizing p53 mutant mice that have recapitulated and informed clinical observations. These studies have demonstrated the p53 contribution, sometimes beneficial and sometimes detrimental, to treatment response in lymphomas, and lung and breast cancers. Further, we examine how p53 mutant mouse models have been used to test the efficacy of p53 reactivation as a therapeutic strategy.

Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines.

Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation, protect cells from apoptosis, and enhance metastasis. In this study, we examined the IGF signaling pathway in two breast cancer cell lines selected for metastatic behavior. LCC6 was selected for growth as an ascites tumor in athymic mice from parental MDA-MB-435 cells (435P). The MDA-231BO cell line was derived from osseous metastases that formed after intracardiac injection of the MDA-MB-231 cell line in athymic mice. Compared to the parental cell lines, IGF-I treatment enhanced IRS-2 phosphorylation over IRS-1 in the metastatic variants. IGF-I stimulated cell migration in the variant cells, but not in the parental cells. To determine the role for IRS-2 in IGF-mediated motility, we transfected MDA-231BO cells with an anti-sense IRS-2 construct. Transfected cells had decreased levels of IRS-2 with diminished IGF-mediated motility and anchorage independent growth when compared to control cells. However, adherence to fibronectin was enhanced in the transfected cells compared to MDA-231BO cells. Our data show that breast cancer cells selected for metastatic behavior in vivo have increased IRS-2 activation and signaling. In these cells, IGF-I enhances cell adhesion and motility suggesting that IRS-2 may mediate these aspects of the malignant phenotype.

Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells.

Akt, when activated by IGF/insulin, can phosphorylate forkhead transcription factors. We undertook this study to determine whether epidermal growth factor (EGF) treatment could produce a signaling cascade resulting in phosphorylation of the forkhead transcription factor FKHR in a breast cancer cell line, MDA-MB-231. After establishing ErbB1, cbl, PI3 kinase and Akt were activated in EGF treated MDA-MB-231, we determined by immunoblot with FKHR antiserum that the electrophoretic mobility of FKHR was retarded after EGF treatment. This mobility retardation was reversible by treatment with alkaline phosphatase, and immunoblot with phospho-Ser256 FKHR antibody further confirmed phosphorylation on an Akt consensus site after EGF treatment. EGF stimulated FKHR phosphorylation was blocked by the PI3 kinase inhibitor LY294002, and the ErbB1 inhibitor AG1478. FKHR immunoblotting after purification of nuclear and cytoplasmic proteins showed that EGF induced a simultaneous increase of FKHR in the cytoplasm and decrease in the nucleus. This finding was confirmed by immunofluorescence staining. Treatment of cells with pharmacological inhibitors of PI3 kinase or ErbB1 blocked this effect. Thus, these results demonstrate the phosphorylation and nuclear exclusion of FKHR after EGF treatment by a PI3 kinase dependent mechanism, and represent the first report of growth factor regulation of endogenous FKHR localization.

Elevated levels of p66 Shc are found in breast cancer cell lines and primary tumors with high metastatic potential.

The adapter molecule Shc has been implicated in specific steps of metastasis. In the current study, we show that the expression and activation of the p66 Shc isoform increased in a highly metastatic variant (F-11) of the human breast cancer cell line MDA-MB-231 compared to the parent cell line, whereas the p46 and p52 Shc isoforms were unchanged. Despite reports that p66 Shc can negatively regulate epidermal growth factor signaling to the mitogen-activated protein kinase pathway, we found no change in epidermal growth factor-stimulated activation of mitogen-activated protein kinase in the F-11 cell line. We determined the level of Shc expression by immunoblot in primary breast cancer specimens obtained from patients with or without axillary node involvement. p66 Shc expression increased in tumors obtained from node-positive patients (Spearman correlation coefficient = 0.43377; P = 0.0058) compared to the node-negative specimens. Furthermore, increasing levels of p66 Shc correlated with an increasing number of positive nodes (P = 0.032). This study shows that p66 Shc expression increased in cultured breast cancer cells selected for metastasis and in primary human breast cancer specimens obtained from patients with lymph node involvement, suggesting a possible role for Shc in human breast cancer metastasis.

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.

FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter.

The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription.

Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo.

Cross-talk between insulin-like growth factor (IGF)- and estrogen receptor (ER)-signaling pathways results in synergistic growth. We show here that estrogen enhances IGF signaling by inducing expression of three key IGF-regulatory molecules, the type 1 IGF receptor (IGFR1) and its downstream signaling molecules, insulin receptor substrate (IRS)-1 and IRS-2. Estrogen induction of IGFR1 and IRS expression resulted in enhanced tyrosine phosphorylation of IRS-1 after IGF-I stimulation, followed by enhanced mitogen-activated protein kinase activation. To examine whether these pathways were similarly activated in vivo, we examined MCF-7 cells grown as xenografts in athymic mice. IRS-1 was expressed at high levels in estrogen-dependent growth of MCF-7 xenografts, but withdrawal of estrogen, which decreased tumor growth, resulted in a dramatic decrease in IRS-1 expression. Finally, we have shown that high IRS-1 expression is an indicator of early disease recurrence in ER-positive human primary breast tumors. Taken together, these data not only reinforce the concept of cross-talk between IGF- and ER-signaling pathways, but indicate that IGF molecules may be critical regulators of estrogen-mediated growth and breast cancer pathogenesis.

Insulin receptor substrate-1 is the predominant signaling molecule activated by insulin-like growth factor-I, insulin, and interleukin-4 in estrogen receptor-positive human breast cancer cells.

Because insulin-like growth factor-I (IGF-I), insulin, and interleukin-4 (IL-4) have known biological effects in breast cancer cells and signal through insulin-receptor substrate (IRS) adaptor proteins, we examined the expression and function of IRS-1 and IRS-2 in breast tumors and cell lines. IRS-1 and IRS-2 were expressed by cell lines and primary breast tumor specimens. IGF-I, insulin, and IL-4 treatment of MCF-7 and ZR-75, and IGF-I treatment of T47-D breast cancer cells, resulted in much greater tyrosine phosphorylation of IRS-1 compared with IRS-2. Furthermore, IGF-I stimulated greater tyrosine phosphorylation of IRS-1 than either insulin or IL-4. IGF-I treatment also enhanced association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with IRS-1 and stimulated increased enzymatic activity compared with IL-4 and insulin in all three cell lines. Similarly, mitogen-activated protein kinase activity was greater in IGF-I-stimulated cells. To determine the functional significance of the activation of these pathways, we inhibited activation of phosphatidylinositol 3-kinase with wortmannin and mitogen-activated protein kinase with PD098059. Both compounds inhibited IGF-stimulated growth, suggesting that both pathways contributed to the mitogenic response to IGF-I. We conclude that IRS-1, and not IRS-2, is the predominant signaling molecule activated by IGF-I, insulin, and IL-4. Furthermore, enhanced tyrosine phosphorylation of IRS-1 by IGF-I, compared with either insulin or IL-4, is associated with greater activation of mitogenic downstream signaling pathways resulting in enhanced cell growth.

The formation of the Medical and Scientific Section of the British Diabetic Association.

James (Jim) Jackson was the first administrative secretary to the Medical and Scientific Section of the British Diabetic Association (BDA). He played an important part in the creation of the Section and its development, bringing order into what had been rather haphazard medical meetings of the Diabetic Association. He has written this history of the Medical and Scientific Section of the BDA, based upon personal recollection with historical data taken from minutes of meetings. He writes: 'The Medical and Scientific Section emerged from a feeling of dissatisfaction among diabetologists and research workers about the post-war activities and aims of the Medical Advisory and Research Grant Committees of the British Diabetic Association. There was also a perceived need to involve physicians in charge of diabetic clinics countrywide more closely in the activities of the Association as a means of increasing its lay membership. This history is based upon personal recollection, with historical data taken from minutes of meetings'. Jim Jackson's history is accompanied by footnotes provided by Dr David Pyke (DAP), former physician in charge of the diabetes service at King's College Hospital, London, and onetime registrar of the Royal College of Physicians of London.

Clinical, motor, and biological correlates of depressive disorders after focal subcortical lesions.

The authors studied depression after focal subcortical lesions (SCLs) in 45 highly selected subjects. Secondary major depression (secondary MD) occurred in 20.0%, depressive disorder NOS (secondary DDNOS) in 4.4%, and secondary dysthymia in 0.0%. secondary MD after SCLs was associated with pallidal lesions (88.9%) and dystonia without geste antagonistique; subjects with secondary DDNOS had nigrotegmental lesions and parkinsonism. Depressive severity after SCLs correlated positively with severity of parkinsonism and dystonia. Pallidal lesions disrupting neurotransmitter systems and pallidothalamic and parietal input to the frontal lobe may lead to secondary MD, whereas nigrotegmental lesions may predispose to secondary MD forme fruste (secondary DDNOS) through disruption of mesocortical frontal or nigrostriatal dopamine tracts. Patients should be closely followed over several years for depression after such lesions, especially when accompanied by parkinsonism or dystonia without geste antagonistique.

Activation of estrogen receptor-mediated gene transcription by IGF-I in human breast cancer cells.

Estrogen and IGF-I are potent mitogens for most breast cancer cell lines, and although their signaling pathways contrast, there is considerable interaction between them. Recent evidence indicating that IGF-I can alter estrogen receptor (ER) action led us to investigate whether an inhibitor of IGF-I action. IGF-binding protein-1 (IGFBP-1), could affect transcriptional activation of ER. First, we confirmed that tamoxifen (TAM) could inhibit IGF-I-mediated proliferation of MCF-7 cells. Although TAM can increase IGFBP-3 expression in MCF-7 cells, and this binding protein has been shown to be able to inhibit IGF action, TAM had no effect on IGF-I-stimulated tyrosine phosphorylation of IGF-I receptor or the downstream signaling molecule, insulin receptor substrate-1. Therefore, to confirm that IGF-I was affecting transcriptional activation of ER, we utilized a gene reporter assay using a single consensus estrogen response element (ERE-tk-luc) upstream of luciferase. As expected, estradiol (E2; 1nM) increased transcriptional activation three- to fivefold from the ERE in three ER-positive breast cancer cell lines (MCF-7, ZR-75 and T47D). A 2.5-to 4-fold increase was also seen with IGF-I (5 nM). TAM (1 microM) effectively blocked activation by E2 and IGF-I, indicating disruption of ER-mediated transcription. As expected, human recombinant IGFBP-1 (80 nM) completely inhibited IGF-I-mediated activation of ER, however, IGFBP-1 also caused a significant decrease in E2-mediated activation. We also noticed that IGF-I increased the activity of all plasmids that we cotransfected including TATA-luc, SV40-luc and pGL Basic. This effect was post-transcriptional, as it was not affected by actinomycin D (2 micrograms/ml), while we were able to completely inhibit E2-mediated transcriptional activation of ERE-tk-luc. Unlike the complete inhibition of ER-mediated transcriptional activation by actinomycin D, IGF-I-mediated transactivation was reduced by only 50%, indicating that the activation by IGF-I represented both transcriptional and post-transcriptional effects. This study confirmed that IGF-I can cause transcriptional activation of endogenous ER in human breast cancel cells, and inhibition of ER action by IGFBP-1 suggests that IGF-1 signaling may be necessary for maximal ER activation.

Insulin-like growth factor binding protein-3 and insulin receptor substrate-1 in breast cancer: correlation with clinical parameters and disease-free survival.

Insulin-like growth factors (IGFs) interact with specific cell surface receptors to mediate cell growth. Intracellular effects of the IGFs are mediated by activation of secondary messenger molecules. One of these proteins, insulin receptor substrate-1 (IRS-1), is phosphorylated after type I IGF receptor activation and has a major role in IGF signaling. Receptor activation also is influenced by high-affinity IGF binding proteins (IGFBPs). In serum, IGFBP-3 is the predominant species. The role of IGFBP-3 in the regulation of breast cancer cell growth is unclear; both growth inhibition and stimulation have been documented in tissue culture systems. To investigate the influence of IGFBP-3 and IRS-1 in breast cancer, we measured levels of these proteins by ELISA and immunoblotting in 195 node-negative primary human breast cancers and compared their levels with known prognostic factors and disease-free survival (DFS). IGFBP-3 levels correlated positively with tumor size (r = 0.27, P < 0.0001) and negatively with estrogen receptor (r = -0.35, P < 0. 0001) and progesterone receptor (r = -0.16, P = 0.021). In contrast, IRS-1 did not correlate with prognostic factors, but higher levels of IRS-1 predicted worse DFS for the subset of patients with tumors

Major depression after left posterior globus pallidus lesions.

We studied subjects with focal subcortical lesions (SCLs) and investigated the frequency of pallidal lesions in secondary major depression (secondary MD) presenting after but not before lesion onset. Forty-five subjects were selected for focal subcortical lesions (SCLs) from 10,000 hospital magnetic resonance imaging (MRI) films. SCLs were ascertained by neuroradiologic criteria. Major depression was ascertained by DSM-III, -III-R, and -IV criteria. We compared subjects with secondary MD to SCL subjects lacking life histories of mood disorders and investigated the lesion distribution among pallidal subregions evident on MRI. We further tested an association between pallidal lesions and secondary MD. Pallidal lesions were present in eight (89%) of nine subjects with secondary MD and 13 (59%) of 22 controls. Left posterior pallidal lesions occurred in four (44%) of the nine subjects with secondary MD and two (9%) of the 22 controls (one-tailed Fisher's exact test p = 0.043). Demographic and other factors did not differ between subjects with secondary MD and controls (using Fisher's exact test or Mann-Whitney U test as statistically appropriate). These data, of small sample size and requiring confirmation, suggest the possibility that abnormal pallidal function may contribute to depressive pathophysiology, perhaps by influencing basal ganglia-thalamocortical mood circuits. Left-lateralized circuits in the posterior pallidum may be of particular relevance. The left pallidal association is compatible with previous findings in poststroke depression. Patients with left pallidal lesions may deserve close monitoring for secondary MD after subcortical lesions.

R.D. Lawrence, H.G. Wells, G.B. Shaw: a vignette.

Among the unpublished autobiographical notes of Dr R.D. Lawrence are numerous references to his contacts and friendship with the author H.G. Wells, who was to become the first President of the (British) Diabetic Association. Wells was responsible for Lawrence being called in for consultation by George Bernard Shaw, a meeting which was to have surprising consequences.

Correlation of insulin-like growth factor-binding protein-3 messenger RNA with protein expression in primary breast cancer tissues: detection of higher levels in tumors with poor prognostic features.

BACKGROUND: The insulin-like growth factor (IGF)-binding proteins (IGFBPs) regulate the actions of the IGFs by influencing interactions between the IGFs and the IGF receptors. IGFBP-3, one of the six known species of IGFBPs, is the predominant IGFBP in serum and is expressed by breast cancer cells. Compared with estrogen receptor (ER)-positive samples, ER-negative breast cancer cell lines and tumors express higher levels of IGFBP-3. Therefore, expression of IGFBP-3 may be relevant in breast cancer biology, although it is unknown whether IGFBP-3 levels correlate with other breast cancer prognostic factors besides ER status. It is also not known how different methods used to measure IGFBP-3 in breast cancer correlate. PURPOSE: We measured IGFBP-3 messenger RNA (mRNA) and protein levels in breast tumors by different methods to test how these methods compare and to investigate the relationship between IGFBP-3 and breast cancer prognostic factors. METHODS: We analyzed 40 human breast tumors and examined IGFBP-3 expression by ligand blot analysis, immunoblot analysis, immunoradiometric assay (IRMA), and ribonuclease protection assay. Another set of 40 breast tumors, selected according to ER and progesterone receptor (PR) status, S phase, and ploidy, was analyzed by IRMA. RESULTS: In 26 (65%) of 40 samples in which RNA could be isolated, IGFBP-3 mRNA levels correlated with IGFBP-3 levels measured by IRMA (two-sided; P = .0001) but not with IGFBP-3 levels measured by ligand blot or immunoblot. Protein levels were highly correlated among all protein assays. Because the IRMA was more sensitive and accurate than the ligand blot and immunoblot assays, we used IRMA to examine IGFBP-3 levels in an additional 20 primary breast tumors with poor prognostic features (ER and PR negativity, high S phase, and aneuploidy) and in 20 tumors with good prognostic factors (opposite features). IGFBP-3 levels were threefold higher in tumors with poor prognostic features (mean +/- standard deviation = 32.8 +/- 25.2 versus 11.8 +/- 9.7 ng/mg; two-sided; P = .003). CONCLUSIONS: These findings suggest that in human breast cancer, IGFBP-3 mRNA and protein levels are correlated and higher levels of IGFBP-3 are detectable in tumors with poor prognostic features. IMPLICATIONS: IGFBP-3 may be involved in the regulation of breast cancer cell growth.

R.D. Lawrence and the formation of the Diabetic Association.

The history of the (British) Diabetic Association is bound up with the life of Dr R.D. Lawrence, its co-founder and first Chairman. Thus the story begins with a review of his early life and experience. He himself committed some memories to paper, in autobiographical context, to which he gave the title A Splendid Life, and this review draws freely on those unpublished notes, with the permission of his sons. Unfortunately, many records, letters and documents were destroyed when the flat of his secretary, the late Miss Lilian Pearce, was damaged by the 1987 hurricane storms, and later vandalized while she was in hospital. Academically brilliant, with his heart set on becoming a surgeon, Robin Lawrence was diagnosed as having diabetes in 1920, at the age of 28. The poor prognosis lead him to set up practice in Florence, where he could at least enjoy the splendours of Italian culture. His health deteriorated badly during the winter of 1922, but alerted to the introduction of insulin therapy by Dr. G.A. Harrison, he returned to King's College Hospital, London and received his first injection of insulin on 31 May 1923. Thereafter, he devoted his life to the understanding of the disease, and the welfare of those affected by it. Together with his patient, the author and scientist H.G. Wells, Robin Lawrence was responsible for the formation of the Diabetic Association, the first patient-oriented association to be established in the United Kingdom. Historical data are taken from early issues of the Diabetic Journal and minutes of meetings to which the author has been given access.

Proliferation of cultured human prostate cancer cells is inhibited by insulin-like growth factor (IGF) binding protein-1: evidence for an IGF-II autocrine growth loop.

In this study, we examined the expression of insulin-like growth factor (IGF) ligands, receptors, (IGFR1, IGFR2), and binding proteins (IGFBPs) in the human prostate cancer cell line DU145, as well as its mitogenic response to the IGFs. Using RNase protection assays, we found expression of IGF-II, IGFR1, and IGFR2 but failed to detect IGF-I messenger RNA. Distinct binding protein species as well as immunoreactive IGF-II were detected in conditioned media using radioligand and immunoblotting assays. Compared with controls, treatment with exogenous IGF-I and IGF-II resulted in stimulation of monolayer and anchorage-independent growth. Recombinant human IGFBP-1, which binds IGF-II with high affinity, inhibited IGF-II-induced monolayer growth and both baseline and IGF-II-induced anchorage-independent growth in this cell line. Our data suggest IGF-II is as an autocrine growth factor in DU145 cells, and that inhibition of IGF-II-dependent growth of human prostate cancer cells may represent a new therapeutic strategy for this disease.