PE-Ynet: a novel attention-based multi-task model for pulmonary embolism detection using CT pulmonary angiography (CTPA) scan images.

Pulmonary Embolism (PE) has diverse manifestations with different etiologies such as venous thromboembolism, septic embolism, and paradoxical embolism. In this study, a novel attention-based multi-task model is proposed for PE segmentation and detection from Computed Tomography Pulmonary Angiography (CTPA) images. A Y-Net architecture is used to implement this model, which facilitates segmentation and classification jointly, improving performance and efficiency. It is leveraged with Multi Head Attention (MHA), which allows the model to focus on important regions of the image while suppressing irrelevant information, improving the accuracy of the segmentation and detection tasks. The proposed PE-YNet model is tested with two public datasets, achieving a maximum mean detection and segmentation accuracy of 99.89% and 99.83%, respectively, on the CAD-PE challenge dataset. Similarly, it also achieves a detection accuracy of 99.75% and a segmentation accuracy of 99.81% on the FUMPE dataset. Additionally, sensitivity analysis also shows a high sensitivity of 0.9885 for the localization error ɛ = 0 for the CAD-PE dataset, demonstrating the model's robustness against false predictions compared to state-of-the-art models. Further, this model also exhibits lower inference time, size, and memory usage compared to representative models. An automated PE-YNet tool can assist physicians with PE diagnosis, treatment, and prognosis monitoring in the clinical management of CoVID-19.

TP53 Status as a Determinant of Pro- vs Anti-Tumorigenic Effects of Estrogen Receptor-Beta in Breast Cancer.

BACKGROUND: Anti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC). METHODS: ESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided. RESULTS: ESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02). CONCLUSIONS: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

MGMT inhibition in ER positive breast cancer leads to CDC2, TOP2A, AURKB, CDC20, KIF20A, Cyclin A2, Cyclin B2, Cyclin D1, ERα and Survivin inhibition and enhances response to temozolomide.

The DNA damage repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) is overexpressed in breast cancer, correlating directly with estrogen receptor (ER) expression and function. In ER negative breast cancer the MGMT promoter is frequently methylated. In ER positive breast cancer MGMT is upregulated and modulates ER function. Here, we evaluate MGMT's role in control of other clinically relevant targets involved in cell cycle regulation during breast cancer oncogenesis. We show that O6-benzylguanine (BG), an MGMT inhibitor decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, cyclin B2, A2, D1, ERα and survivin and induces c-PARP and p21 and sensitizes ER positive breast cancer to temozolomide (TMZ). Further, siRNA inhibition of MGMT inhibits CDC2, TOP2A, AURKB, KIF20A, Cyclin B2, A2 and survivin and induces p21. Combination of BG+TMZ decreases CDC2, CDC20, TOP2A, AURKB, KIF20A, Cyclin A2, B2, D1, ERα and survivin. Temozolomide alone inhibits MGMT expression in a dose and time dependent manner and increases p21 and cytochrome c. Temozolomide inhibits transcription of TOP2A, AURKB, KIF20A and does not have any effect on CDC2 and CDC20 and induces p21. BG+/-TMZ inhibits breast cancer growth. In our orthotopic ER positive breast cancer xenografts, BG+/-TMZ decreases ki-67, CDC2, CDC20, TOP2A, AURKB and induces p21 expression. In the same model, BG+TMZ combination inhibits breast tumor growth in vivo compared to single agent (TMZ or BG) or control. Our results show that MGMT inhibition is relevant for inhibition of multiple downstream targets involved in tumorigenesis. We also show that MGMT inhibition increases ER positive breast cancer sensitivity to alkylator based chemotherapy.

Racial Disparity Among Women Diagnosed With Invasive Breast Cancer in a Large Integrated Health System.

PURPOSE: Reasons for the well-described disparity in outcomes between African American (AA) and non-Hispanic white (NHW) women with invasive breast cancer are unclear, making it difficult to identify solutions. This study examined the effects of demographics, biomarkers, tumor characteristics, cancer stage, morphology, and treatment variables on overall and cancer-free survival in these patient populations. METHODS: We retrospectively reviewed data for 6231 patients diagnosed with invasive breast cancer throughout an integrated health system from January 2006 through March 2015. Included for analysis were 5023 NHW and 413 AA women. All category and continuous variables in the study were described in the two groups using appropriate statistics. Kaplan-Meier method of survival with log-rank test was used to compare the two racial groups (NHW and AA). Cox proportional hazards regression was used to find hazard ratios for the predictors of survival and recurrence-free survival probability. Propensity probability match method (1:1) was used to match 319 NSW women to 319 similar AA women. Matching was done using all significant predictors, including demographic variables. RESULTS: Compared to NHW women, AA women presented with invasive breast cancer at a younger age (P<0.001) and had a higher proportion of stage IV cancers (P<0.001), which were more often infiltrating ductal carcinoma (P<0.003) and poorly differentiated (P<0.001). Within 10-year follow-up, AA women had shorter overall and recurrence-free survival (log-rank P<0.001), were 1.4 times more likely to die (P=0.009), and were twice as likely to have recurrence (P<0.001) than NHW women. In the matched groups, overall survival was similar for AA and NHW (log-rank P=0.0793); however, recurrence-free survival was higher in NHW than in AA women (P=0.047). CONCLUSIONS: When presenting characteristics of AA and NHW women with invasive breast cancer are matched, disparity in overall mortality and rate of recurrence appears to be reduced or perhaps eliminated, suggesting invasive breast cancers in AA and NHW women respond similarly to treatment. Further study is needed to explore the true effect of biological factors; however, rectifying delivery of and access to care might be expected to mitigate, in large part, the racial disparity currently seen in breast cancer outcomes.

Application of microbial transglutaminase in meat foods: A review.

Microbial transglutaminase (MTG) is an enzyme isolated from a variant of Streptomyces mobaraensis that forms covalent cross-links between protein molecules. Studies are being conducted since last two decades on utilization of MTG in meat foods to improve their characteristics, such as gelation, water-binding, emulsion stability, purge loss, cooking loss, etc. MTG is one of the important topics of interest in meat processing industry due to its advantages in practical utilization and commercial exploitation. This review will discuss about the overall applications of MTG in manipulating the functional properties of meat and meat products by means of various processes such as restructuring, value addition, etc.

Factors influencing meat emulsion properties and product texture: A review.

Emulsion-based meat products play an important role in modern meat industry. Though meat batters have been prepared traditionally since long back in the history, the scientific principles and the knowhow are significantly important in the case of commercial products. In India, the market for emulsion meat products is gaining importance in the recent years and the native producers are in critical need for the scientific basis of production of emulsion meat products with better yield, good sensory qualities and nutrition. Hence, this review will throw light on some of the important factors which influence the properties of meat emulsion such as stability, structure, etc. and the product texture and yield as the revealed by past researches which will be useful to the meat processors in their practical application in preparing meat emulsion products.

T cell recall response of two hypothetical proteins (Rv2251 and Rv2721c) from Mycobacterium tuberculosis in healthy household contacts of TB - Possible subunit vaccine candidates.

The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) encourages the need for new vaccine candidates against TB. Antigen specific cellular immune response is often considered imperative during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine development. Here, we investigated the phenotypic and functional profiles of two novel mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow cytometry. Healthy household contacts of TB (latent/HHC) and active pulmonary TB (PTB) patients were recruited to investigate the difference in antigen specific T cell recall response. These two antigens induced expansion of CD45RA- CCR7+ central memory subtypes and CD45RA- CCR7- effector memory cells in latent population which suggests their possible association with HHC. Rv2251 and Rv2721c antigen specific IFN-γ, TNF-α and IL-2 response was also significantly high in HHC when compared to the PTB (p < 0.005, p < 0.05 and p < 0.05 respectively). The frequency of multifunctional T cells also was high in HHC compared to the PTB with statistical significance only for the antigen Rv2251. Often, the dominant Th1 immune response in HHC is correlated with the protection against the active TB disease. Collectively, we report the first insights into Rv2251 and Rv2721c antigen specific immune response in human donors of TB and provide the immunologic rationale for selecting them for vaccine development against TB.

Segmentation and classification of bright lesions to diagnose diabetic retinopathy in retinal images.

In view of predicting bright lesions such as hard exudates, cotton wool spots, and drusen in retinal images, three different segmentation techniques have been proposed and their effectiveness is compared with existing segmentation techniques. The benchmark images with annotations present in the structured analysis of the retina (STARE) database is considered for testing the proposed techniques. The proposed segmentation techniques such as region growing (RG), region growing with background correction (RGWBC), and adaptive region growing with background correction (ARGWBC) have been used, and the effectiveness of the algorithms is compared with existing fuzzy-based techniques. Images of eight categories of various annotations and 10 images in each category have been used to test the consistency of the proposed algorithms. Among the proposed techniques, ARGWBC has been identified to be the best method for segmenting the bright lesions based on its sensitivity, specificity, and accuracy. Fifteen different features are extracted from retinal images for the purpose of identification and classification of bright lesions. Feedforward backpropagation neural network (FFBPNN) and pattern recognition neural network (PRNN) are used for the classification of normal/abnormal images. Probabilistic neural network (PNN), radial basis exact fit (RBE), radial basis fewer neurons (RB), and FFBPNN are used for further bright lesion classification and achieve 100% accuracy.

MGMT inhibition suppresses survivin expression in pancreatic cancer.

OBJECTIVES: Survivin, an antiapoptotic gene inhibited by p53, is overexpressed in human cancers and correlates with chemotherapy resistance. Here, we investigated the mutual regulatory mechanism between MGMT (O-methylguanine DNA methyltransferase) and survivin. METHODS: This study used standard techniques for protein and messenger RNA levels, promoter activity, protein-DNA interaction, cell viability, and correlative animal model. RESULTS: O-benzylguanine (BG), a potent inhibitor of MGMT (a DNA repair protein), curtails the expression of survivin in pancreatic cancer. Silencing MGMT by small interfering RNA down-regulates survivin transcription. p53 inhibition enhances MGMT and survivin expressions. When p53 was silenced, BG-induced MGMT inhibition was not associated with the down-regulation of survivin, underscoring the regulatory role of p53 in the MGMT-survivin axis. O-benzylguanine inhibits survivin and PCNA (proliferating cell nuclear antigen) at messenger RNA and protein levels in PANC-1 and L3.6pl cells and decreases survivin promoter activity via increased p53 recruitment to the survivin promoter. In orthotopic pancreatic xenografts established in nude mice, BG ± gemcitabine (GEM) decrease survivin expression in tumor tissue; protein levels and immunohistochemistry show significant decrease in survivin and PCNA levels, which correlate with increased sensitivity to GEM. CONCLUSIONS: MGMT inhibition is associated with decrease in survivin expression and increase in sensitivity to GEM in pancreatic cancer.

MGMT inhibition restores ERα functional sensitivity to antiestrogen therapy.

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.

Development and Validation of a UV Spectrophotometric Method for the Simultaneous Estimation of Eprosartan Mesylate and Hydrochlorothiazide in Bulk and Formulations.

A simple, efficient, precise and accurate absorbance ratio method have been developed for the estimation of eprosartan mesylate and hydrochlorothiazide in pure and in fixed dose combination. In this method, UV spectra of eprosartan mesylate and hydrochlorothiazide were overlayed which involves the formation of Q-absorbance equation at 249.1 nm (isobestic point) and 274.5 nm, the max of hydrochlorothiazide. Both the drugs obeyed Beers law in the concentration range of 6-36 µg/ml and 1-10 µg/ml for eprosartan mesylate and hydrochlorothiazide, respectively. The accuracy of the method was determined by recovery studies and was found to be in the range of 102.29-103.10% and 99.52-101.60% for eprosartan mesylate and hydrochlorothiazide, respectively. The method was validated as per ICH guidelines and statistically. The method showed good reproducibility and recovery with % RSD less than 2. The method was found to be simple, economic, accurate and reproducible and can be used for routine analysis of eprosartan mesylate and hydrochlorothiazide in pure and in fixed dose combinations.

Mechanisms of estrogen receptor antagonism toward p53 and its implications in breast cancer therapeutic response and stem cell regulation.

Estrogen receptor alpha (ERalpha) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERalpha binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERalpha suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERalpha represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor p21(Waf1/Cip1) (CDKN1A) gene, a prototypic transcriptional target of p53. While 17beta-estradiol (E2) enhanced ERalpha binding to p53 and inhibited p21 transcription, antiestrogens decreased ERalpha recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERalpha target gene, pS2/TFF1. These results suggest that ERalpha uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERalpha binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response.

Levetiracetam enhances p53-mediated MGMT inhibition and sensitizes glioblastoma cells to temozolomide.

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an unrecognized impact in modulating O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions. In vitro, when used at concentrations within the human therapeutic range for seizure prophylaxis, LEV decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation analysis reveals that LEV enhances p53 binding on the MGMT promoter by recruiting the mSin3A/histone deacetylase 1 (HDAC1) corepressor complex. However, LEV does not exert any MGMT inhibitory activity when the expression of either p53, mSin3A, or HDAC1 is abrogated. LEV inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide. In 4 newly diagnosed patients who had 2 craniotomies 7-14 days apart, prior to the initiation of any tumor-specific treatment, samples obtained before and after LEV treatment showed the inhibition of MGMT expression. Our results suggest that the choice of AED in patients with malignant gliomas may have an unrecognized impact in clinical practice and research trial design.

Blockade of MGMT expression by O6 benzyl guanine leads to inhibition of pancreatic cancer growth and induction of apoptosis.

PURPOSE: We sought to determine whether administration of a MGMT blocker, O(6)-benzyl guanine (O(6)BG), at an optimal biological dose alone or in combination with gemcitabine inhibits human pancreatic cancer cell growth. EXPERIMENTAL DESIGN: Human pancreatic cancer L3.6pl and PANC1 cells were treated with O(6)BG, either alone or in combination with gemcitabine, and the therapeutic efficacy and biological activity of these drug combinations were investigated. RESULTS: O(6)BG sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O(6)BG. In sharp contrast, protein expression and mRNA message of p21(cip1) were significantly increased. Interestingly, O(6)BG increases p53-mediated p21(cip1) transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21(cip1) and a decrease in cyclin transcription are p53 dependent. The volume of pancreatic tumors was reduced by 27% in mice treated with gemcitabine alone, by 47% in those treated with O(6)BG alone, and by 65% in those mice given combination. Immunohistochemical analysis showed that O(6)BG inhibited expression of MGMT and cyclins, and increased expression of p21(cip1). Furthermore, there was a significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis. CONCLUSIONS: Collectively, our results show that decreased MGMT expression is correlated with p53 activation, and significantly reduced primary pancreatic tumor growth. These findings suggest that O(6)BG either alone or in combination with gemcitabine may provide a novel and effective approach for the treatment of human pancreatic cancer.

Neuroacanthocytosis: a rare inherited movement disorder.

The chorea-acanthocytosis syndrome (CHAC) is a rare disorder beginning in late adolescent or adult life in association with acanthocytosis, a normal lipid profile and characterized by progressive neurological disease. The inheritance is usually autosomal recessive, although apparent sporadic and autosomal dominant instances are also known. We report here a young man who presented with choreo-athetoid movement, dystonia, tics, symmetrical axonal polyneuropathy with normal cognitive function. The subsequent peripheral blood film reveals acanthocytes > 5%. Diagnosis of neuroacanthocytosis was made.

Estrogen-mediated downregulation of CD24 in breast cancer cells.

We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ER alpha negative MDA-MB-231 cells suggesting that ER alpha was necessary. This was further confirmed by ER alpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ER alpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ER alpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ER alpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ER alpha and histone deacetylases.

Estrogen receptor alpha inhibits p53-mediated transcriptional repression: implications for the regulation of apoptosis.

Estrogen receptor alpha (ERalpha) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERalpha binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERalpha inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERalpha interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERalpha binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERalpha is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERalpha to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERalpha to repress p53-mediated transcriptional activation. Further, RNA interference-mediated knockdown of ERalpha resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERalpha opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer.

Estrogen receptor-alpha binds p53 tumor suppressor protein directly and represses its function.

Estrogen receptor-alpha (ERalpha) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERalpha and p53. Ionizing radiation together with ERalpha knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the pro-proliferative role of ERalpha.

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Physiological and chemical inducers of tissue factor pathway inhibitor-2 in human glioma cells.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extracellular matrix, is expressed in high amounts in low-grade, non-invasive glioma cells but in low amounts in high-grade, highly invasive glioma cells. Overexpression of TFPI-2 by highly invasive glioma cells reduces their invasiveness and thus may be useful in cancer therapy. The mechanisms underlying the transcriptional regulation of TFPI-2 are not well elucidated. We previously reported that the -312 to +1 region of TFPI-2 was critical for the minimal, inducible regulation of TFPI-2 in gliomas. This region harbors sites for several transcription factors, including SP1 (-192 to -183 and -135 to -128), AP-1 (-310 to -300, -213 to -204, and -163 to -154), NF-kappaB (-229 to -221), an NF-kappaB-like site (-291 to -281), and Lyf-1 (-260 to -252). Here we transiently transfected low-grade Hs683 glioma cells with mutant constructs to clarify the role of these transcription factors in TFPI-2 regulation. Addition of phorbol 12-myristate 13-acetate, 1,2-diacyl-sn-glycerol, IFN-gamma, or IFN-alpha induced the expression of TFPI-2 wild-type promoter construct as well as TFPI-2 protein and mRNA in Hs683 cells. Mutations at either of two AP-1 sites (-310 to -300 and -163 to -154) or either of two SP1 sites (-192 to -183 and -135 to -128) resulted in reduced TFPI-2 activity, regardless of the presence of stimulator compounds, and reduction in DNA-protein binding (by electrophoretic mobility shift assay).