Presence Of Wt Research Articles

The D75N and P161S Mutations in the C0-C2 Fragment of cMyBP-C Associated with Hypertrophic Cardiomyopathy Disturb the Thin Filament Activation, Nucleotide Exchange in Myosin, and Actin-Myosin Interaction.

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

The effect of different clay types on the barrier properties of PLA/PBAT/clay bio-nanocomposite packaging films

The aim of this study was to develop alternative bio-nanocomposite film to Polyethylene (PE) film from poly(lactic acid) (PLA) and poly(butylene adipate-co-terephthalate) (PBAT) blends and different clay types for flexible packaging industry. Poly(lactic acid)/poly(butylene adipate-co-terephthalate) (PLA/PBAT) bio-blend films were prepared by using a chain extender. Bio-nanocomposite films were processed on a twin-screw extrusion and blown film extruder by wt.%3 loading the different montmorillonite (MMT), Sepiolite, and Bentonite types in presence of a polymeric epoxy-based chain extender plasticizer (CE; Joncryl® ADR 4468). All films were characterized in terms of thermal, chemically, physically, optical, mechanical, sealing and barrier properties. In conclusion, all analyzing results showed good agreement with PLA/PBAT in the presence of a polymeric epoxy-based chain extender. The main outcome of adding clay types to PLA/PBAT bio-blends is to obtain eco-friendly, improved barrier properties compared to PE film structure, which has inherently poor oxygen permeability. As a result of all studies, the closest alternative bio-blend recipes to PE film structure with improved barrier properties were obtained with composite of wt.%25 PLA + %75 PBAT in the presence of wt.%1 plasticizer and %3 High Purity Sepiolite clay (HPS).

Abstract 1409: RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer

Abstract Background: African American men (AAs) have the highest incidence of prostate cancer (PC) and often develop therapeutic resistance leading to high mortality. The androgen receptor (AR) is paramount in the growth and progression of PC, and insufficient blockade of AR leads to therapeutic resistance. Our previous work shows that CYP3A5 facilitates the activation of AR, promoting the transcription of genes supporting PC growth. CYP3A5 is polymorphic, and the wild-type (wt; *1) variant encoding the full-length active protein is expressed in 73% of AAs and only in 5% of non-Hispanic White Americans (NHWA). The race-linked expression of wt-CYP3A5*1 in AAs can lead to hyperactive AR contributing to therapeutic resistance. We performed an RNA sequencing study utilizing samples from AAs and NWHA patients to understand the allelic influence of CYP3A5 on PC severity. Methods: Illumina RNA sequencing was performed using RNA from 13 AA and 12 NHWA prostatic adenocarcinomas. CYP3A5 genotyping separated them into two groups: a) carrying wt-CYP3A5 (*1/*1 and *1/*3; N=14); and b) mutant CYP3A5 (*3/*3; N=11) variants. The samples’ ancestry was confirmed using markers previously described. Differentially expressed genes (DEGs) between wt-*1 and mutant-*3 CYP3A5 carrying groups were identified using DESeq2, at a false discovery rate of adjusted p < 0.05. Alternately we also used the Kallisto method for quantifying the abundances of the transcript from RNA seq data. Results: Aligned RNA reads with the human genome using DESeq2 resulted in the identification of 30 DEGs between the two groups, of these, 14 genes are known to be dysregulated in PC and promote aggressive disease (SEMG2, ZNRF3, CCL18, U3, MT-ND6, SCARNA9, NRCAM, LINC00853, TCF4, SLC38A6, HHLA3, DECR1, THTPA, and NEFH). Aberrant TCF4 along with ZNRF3 is known to promote PC growth via Wnt-beta catenin signaling. SLC38A6 is a glutamine transporter and is often upregulated in PC to promote glutamine uptake in an AR-dependent manner. NEFH is an intermediate filament protein that is downregulated in PC metastasis. Kallisto method identified 84 DEGs, 11 of those genes overlap with the previous DFGs identified using the transcriptome alignment method. The additional DFGs identified with the Kallisto method include aberrant expression of multiple genes (MT-CO1/2, MT-ND1/6) involved in mitochondrial dysfunction known to contribute towards cell growth and tumorigenesis beyond the Warburg effect. The wt-*1 CYP3A5 expressing group also shows increased expression of several pseudogenes, dysregulation of pseudogenes has been observed in several cancers and can act as a master regulator for gene expression, promoting tumorigenesis. Conclusion: The presence of wt-*1 CYP3A5 may be one of the factors leading to aggressive PC in AAs as it shows differential expression of several genes known to accelerate PC growth. Citation Format: Jeetesh Sharma, Richard Tillett, Shirley Shen, Jabril Johnson, Rick A. Kittles, Mohammad Saleem Bhat, Oscar Goodman Jr, Edwin C. Oh, Ranjana Mitra. RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1409.

Elucidating the Interaction and Stability of Withanone and Withaferin‐A with Human Serum Albumin, Lysozyme and Hemoglobin Using Computational Biophysical Modeling

AbstractWithania somnifera consists of various bioactive compounds, of which withanone (WT−N) and withaferin‐A (WT−A) are known to have many therapeutic properties. To understand drug metabolism and transportation methods, binding of endogenous and exogenous ingredients with transport proteins is of utmost importance. Therefore, to perceive the molecular recognition mechanism of WT−N and WT−A, the association of human serum albumin, human hemoglobin and human lysozyme with WT−N and WT−A were examined, using various in silico techniques. For our study, we have employed both quantum mechanics and molecular mechanics approaches. Resultantly, drug‐like properties and reactivity were calculated with WT−N and WT−A being the most reactive among all the withanolides. The protein HSA in presence of WT−N showed more stability than that of WT−A. However, HL and Hb showed better stability with WT−A as compared to WT−N. Several non‐covalent interactions contributed the utmost part in the binding of WT−N and WT−A with the proteins. The MD simulation studies also showed evidence in the conformational changes and other dynamical properties of the three proteins with both the ligands. Finally, MM‐PBSA results confirmed the free binding energy of the complexes. Our study has provided a substantial recognition of bioactive molecules WT−N and WT−A binding with the carrier proteins, which can significantly affect the pharmacokinetics of these molecules.

Effect of Weeping Teats on Intramammary Infection and Somatic Cell Score in Dairy Goats.

Mastitis is the most common disease affecting dairy goats and causing economic losses. Although it is accepted that increased somatic cell count (SCC) is mainly a response to infection, its reliability for subclinical mastitis detection in goats is controversial. Indeed, many physiological and extrinsic variables can increase SCC, including breed, parity, age, stage of lactation, seasonal variations, and milking methods. In some animals, milk-secreting tissue is present in the wall of the teat and, in some instances, milk can filter through pores in the skin to the udder surface. This condition is known as “weeping teat” (WT). In these animals, mammary tissue might be prone to develop bacterial infections, although limited information is provided. Weeping teat seems to have a genetic background and is reported to be especially found in goat breeds selected for high milk production. Moreover, it is observed a genetic correlation between WT and decreased milk yield as well as increased somatic cell scores (SCS). Since information on this topic is very limited, this study aimed at investigating any possible relationship between WT, high SCC, and the presence of bacteria in goat milk. Alpine goat farms in Northern Italy were selected based on the presence of WT. Each herd was divided into two age-matched groups, identified as case (WT+) and control (WT–). Half-udder milk samples were collected aseptically at three timepoints; bacteriological analysis was performed, and SCC were determined and transformed in SCS. There was a positive association between SCS and the presence of bacteria in milk (P = 0.037) overall, whereas WT udder defect was associated with positive bacterial culture in just one herd (P = 0.053). Thus, this herd was further investigated, repeating the sampling and the analysis on the following year. The positive association between high SCS and the presence of bacteria in milk was then confirmed (P = 0.007), whereas no association with WT condition was found. These results indicate that WT defect is usually unrelated to both the outcome of milk bacterial culture and SCS. As a side outcome, we could confirm the role of bacterial infection in increasing SCS.

Combination of fulvestrant and chemotherapy in ESR1 Y537S mutant breast cancer cells and potential synergy mechanism related to p53 wildtype.

1065 Background: The acquisition of ligand-independent ESR1 mutations during endocrine therapy in metastatic ER+ breast cancer is a common mechanism of resistance to endocrine treatment, particularly aromatase inhibitors, and found in more than 30% of patients with metastatic ER+ breast cancer. Our recent work showed that the ER mutations confer resistance to currently available endocrine treatments while promoting an aberrant ER transcriptional activity that drives metastases. These results underscore the importance of targeting the ESR1 mutations even after the development of endocrine resistance. We hypothesized that during chemotherapy treatment, the ESR1 mutations remain an important driver of tumor growth and metastases and therefore drugs targeting the ESR1 mutation could enhance the efficacy of chemotherapy. In this study we investigated the combination of chemotherapy with the selective estrogen receptor degrader in the presence of WT and mutant ER. Methods: We performed synergy studies testing the combination of fulvestrant with chemotherapy treatments commonly used in ER+ metastatic breast cancer including 5FU (representing capecitabine), adriamycin and paclitaxel using MCF7 and T47D breast cancer cell lines engineered to express doxycycline inducible Y537S-ESR1 mutation. Results: We found that in MCF7 cells the combination of chemotherapy and fulvestrant was synergistic and the synergy was augmented with the induction of the Y537S mutation. In contrast, there was no synergy in T47D cells that harbor a P53 mutation. We confirmed that the synergistic activity of fulvestrant with chemotherapy is dependent on P53 by generating P53 knock-out MCF7 cells using CRISPR-cas9. Additionally, cell cycle and apoptosis analyses showed that the synergistic activity was mainly due to increased effects on G1 arrest rather than apoptosis. Conclusions: Our study indicates that chemotherapy and fulvestrant are synergistic in ER+ breast cancer and the synergy is increased in the presence of the Y537S ESR1 mutation and is dependent on P53 activity. These results support a clinical trial testing the addition of fulvestrant or other novel selective estrogen receptor degraders in patients with metastatic ER+ breast cancer who are starting chemotherapy treatment.

Cholera toxin requires microbiota metabolites short-chain fatty acids for its mucosal adjuvant activity

Abstract Cholera toxin (CT) is a potent mucosal adjuvant, however, the mechanisms by which CT induces both mucosal and systemic immune responses are still not completely understood. It has been shown recently that gut microbiota is critical in inducing immune response by CT, however, the mechanisms involved remain largely unknown. We demonstrated in this study that gut microbiota metabolites short-chain fatty acids (SCFA), which are solely metabolic products of gut microbiota from dietary fiber, are required for CT in inducing antigen-specific IgG and IgA responses after oral immunization. Depleting of gut bacteria by antibiotics significantly decreased mucosal and systemic IgG and IgA responses in mice orally immunized with ovalbumin (OVA) and CT. Feeding SCFA acetate and butyrate rescued antibody responses in antibiotics-treated WT mice. Furthermore, deficiency of GPR43, a SCFA binding receptor, impaired CT’s adjuvant activity. Acetate and butyrate did not directly act on B cells to promote IgG production, but promoted B cell IgG production and plasma B cell differentiation related genes in the presence of WT but not GPR43−/− DC. Furthermore, SCFA increased IgG production by both WT and GPR43−/− B cells when cultured with WT DC, indicating a crucial role for DC expression of GPR43 in SCFA induction of B cell IgG production. Mechanistically, SCFA promoted DC expressions of BAFF, a B cell activating factor, and Aldh1a2, an enzyme promoting production of retinoic acid (RA) from Vitamin A. Blockade either RA or BAFF signaling significantly inhibited SCFA induced B-cell IgG production. Therefore, our study indicated a critical role of gut microbiota and their metabolites SCFA in promoting the mucosal adjuvant activity of CT through partially GPR43.

The increase in positively charged residues in cecropin D-like Galleria mellonella favors its interaction with membrane models that imitate bacterial membranes

A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and two cationic peptides derived from its sequence, ΔM1 (+5) and ΔM2 (+9) is reported in this work. The influence of charge on the interactions between peptides and membranes and its effect on phase were studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that ΔM2 peptide showed the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol (PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to determine lipid segregation in the presence of peptides. When WT and ΔM1 bound to model membrane containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, ΔM2 peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also suggested that the presence of ΔM1 and ΔM2 peptides increased lipid carbonyl group hydration in DMPG membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in the presence of WT and ΔM1 peptides in DMPC/DMPG. Overall the increase in positively charged residues favors the interaction of the peptides with the negatively charged membrane and its perturbation.

The impact of the endothelial protein C receptor on thrombin generation and clot lysis

IntroductionWhen thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC. MethodsTo determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells). ResultsIn the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation. ConclusionsWe demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis.

Inflammatory chemokine CCL3 Leads to enhanced antitumor development in the draining lymph node (TUM10P.1053)

Abstract CCL3 has been shown to be important in orchestrating cellular contacts in vaccinated lymph nodes. Moreover, dendritic cells can mediate both tumor tolerance and anti-tumor immunity in some tumor models. We hypothesize that incorporating CCL3 into tumor vaccines can enhance anti-tumor immunity. We inoculated naïve recipient mice with either WT or CCL3-secreting colon tumors. CCL3-secreting tumors showed enhanced antitumor immunity and suppressed growth kinetics in immunocompetent mice versus WT-tumors. Relative to non-injected controls or WT-injected tumors, 3 days after injections CCL3-secreting tumors revealed an increase of CD4+, CD8+, and CD19+ lymphocyte in the draining lymph node. Concurrently we conducted in vitro BMDCs experiments to assess the effects the tumor microenvironment (+/- CCL3) has on the capacity of DCs to upregulate T cell activation and adhesion molecules. BMDCs differentiated in the presence of CCL3 showed a greater capacity to upregulation CD86, ICAM1, CD11b, and CCR7 before and after LPS stimulation. Moreover, BMDCs cultured in the presence of WT and CCL3-secreting tumor supernatants revealed an increase in CD86, MHCII, and CCR7 before and after LPS stimulation. Together these data reveal that CCL3 in the tumor microenvironments leads to a reduction in tumor growth and early lymphocyte accumulation in the DLN, which may be a result of an enhanced capacity for DCs to home to the DLN and activate antitumor T cells.

Cell‐intrinsic and ‐extrinsic control of Treg‐cell homeostasis and function revealed by induced CD28 deletion

While the requirement for CD28 and its ligands for the generation and function of "natural" (n)Treg cells is well established, it has not been possible yet to investigate cell-intrinsic effects after interrupted CD28 expression. Here, we demonstrate a selective loss of Treg cells after disruption of the CD28 gene. The decline in Treg-cell number was accompanied by reduced homeostatic proliferation, probably due to lack of costimulation during self-antigen recognition, and by impaired Treg-cell function including downregulation of CTLA-4. The decline in Treg-cell number was unaffected by thymectomy or by the presence of CD28 expressing T cells within the same animal, indicating that impairment of peripheral homeostasis and function of nTreg cells by CD28 deletion is cell-intrinsic. In contrast, downregulation of CD25, the α chain of the IL-2R, did not occur in the presence of WT T cells, indicating that its expression does not depend on CD28 signals in cis.

Stability of the transthyretin molecule as a key factor in the interaction with a-beta peptide--relevance in Alzheimer's disease.

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

T Cell-Intrinsic and -Extrinsic Contributions of the IFNAR/STAT1-Axis to Thymocyte Survival

STAT1 is an essential part of interferon signaling, and STAT1-deficiency results in heightened susceptibility to infections or autoimmunity in both mice and humans. Here we report that mice lacking the IFNα/β-receptor (IFNAR1) or STAT1 display impaired deletion of autoreactive CD4+CD8+-T-cells. Strikingly, co-existence of WT T cells restored thymic elimination of self-reactive STAT1-deficient CD4+CD8+-T cells. Analysis of STAT1-deficient thymocytes further revealed reduced Bim expression, which was restored in the presence of WT T cells. These results indicate that type I interferons and STAT1 play an important role in the survival of MHC class I-restricted T cells in a T cell intrinsic and non-cell intrinsic manner that involves regulation of Bim expression through feedback provided by mature STAT1-competent T cells.

Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer

MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in Escherichia coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.

Effect of single-nucleotide polymorphisms in the CYP19 gene on response to Letrozole among breast cancer patients

22096 Background: Letrozole's effect in metastatic breast cancer (BC) patients was shown to be associated with a single nucleotide polymorphism (SNP) in the CYP19 aromatase gene. Two CYP19 polymorphisms; the 3'UTR rs10046 and IVS4(tct)± significantly correlate with serum estrogen levels in postmenopausal women. We evaluated their impact on response among BC patients treated with Letrozole. Methods: 70 BC patients treated with Letrozole with clinically evaluable disease (local stage III in 3, and metastatic in 67) were included. 65/67 metastatic patients, were treated with Tamoxifen before Letrozol (37-adjuvant, 28-first line for metastatic disease). DNA from whole blood was genotyped using DHLPC. Presence of WT or SNP was recorded. Correlation with tumor response (progressive, stable or responsive) in all patients and time to progression (TTP) from date of Letrozole start to progression were analyzed in 67/70 patients with metastatic disease. Results: Median age at diagnosis was 52 (range: 27–86). In 66, tumor was positive for estrogen receptor, in 2 it was negative and in additional 2 unknown. 28/70 (40%) carried the 3'UTR rs10046 wild type allele (WT). In the IVS4(tct)±, 3 genotypes, homozygous IVS4(tct)+, heterozygous IVS4(tct)± and homozygous IVS(tct)− were recorded with corresponding frequencies of 22/68 (32%), 30/68 (44%) and 15/68 (22%). These genotypes were not significantly associated with tumor response. However, median TTP was not reached at 30.4 months among carriers of IVS4 (tct)−/− allele compared with 14 and 12.7 months in heterozygots and homozygous (tct)+/+ respectively (p=0.02). Conclusion: homozygous delition of the (tct) at IVS4(tct)± of CYP19 is associated with longer TTP in patients with metastatic BC treated with Letrozole. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis

Transcriptional Regulation of ATP2C1 Gene by Sp1 and YY1 and Reduced Function of its Promoter in Hailey–Hailey Disease Keratinocytes

Hailey-Hailey disease (HHD) is a blistering skin disease caused by malfunction of the Ca2+-dependent ATPase, ATP2C1. In this study, key regulatory regions necessary for the expression of the gene encoding human ATP2C1 were investigated. The transient reporter assay demonstrated that region +21/+57 was necessary for activation of the ATP2C1 promoter, and the electrophoretic mobility shift assay demonstrated that the region was recognized by the transcription factors, Sp1 and YY1. In accordance with this result, when Sp1 or YY1 was overexpressed in keratinocytes, an obvious increase in ATP2C1 promoter activity was observed, which was in contrast with the case where a mutant promoter lacking the binding sites for Sp1 and YY1 was used as the reporter. Ca2+-stimulation signal increased nuclear Sp1 proteins and ATP2C1 mRNA levels in normal keratinocytes. In contrast, both these increases were suppressed in keratinocytes from HHD patients. These results indicate that Sp1 and YY1 transactivate the human ATP2C1 promoter via cis-enhancing elements and that incomplete upregulation of ATP2C1 transcription contributes to the keratinocyte-specific pathogenesis of HHD. This is a report describing the regulation of the expression of ATP2C1.

Comparison of p53 antibody status and carcinoembryonic antigen (CEA) levels and their clinical impact as diagnostic markers in colorectal cancer patients

9725 Background: The purpose of this study was to evaluate the clinical impact of p53 antibodies, suggested to be associated with advanced tumor state and poor survival rate. Methods: We analyzed the frequency of circulating anti-p53 antibodies, levels of free p53 protein, and concentration of CEA in sera of colorectal cancer patients (n=48, ELISA) as well as expression of p53/CEA in the tumor (immunohistology) and correlated with the UICC stage. Results: 39.6% of all tested patients (n=19/48) expressed wild-type (wt) p53-specific-IgG-antibodies: 26% in UICC I (n=5/19), 16% in UICC II (n=3/19), 21% in UICC III (n=4/19), and 37% in UICC IV (n=7/19). 31.3% (n=15/48) showed a negative response to p53 and 29.2% (n=14/48) were within the critical range. In addition, similar levels of p53-IgG1/IgG2-subtypes were observed in patients with p53 antibodies (ELISA). No correlation was found between levels of free p53 protein and antibody levels. Immunohistologically 62.5% of the tumors (n=10/16) overexpressed p53 (clone DO-7) independent of UICC stage. The presence of wt p53-IgG-antibodies was correlated with strong p53 staining within the tumor. In addition, 29.2% (n=14/48) of the patients expressed high CEA levels depending on their UICC stage (n=0/6 UICC I, n=0/12 UICC II, n=2/9 UICC III, and n=12/21 UICC IV, ELISA). CEA expression within the tumor and UICC stage was strongly correlated (75%, n=12/16). Five different point mutations at 5 different positions of wt p53 sequence (4 in DNA-binding core domain) were observed in tumors (n=16, RT-PCR), indicating no association with the p53 humoral response and UICC stage. Conclusions: This study suggests that a humoral response to p53 is strongly correlated with intracellular accumulation of p53 within the tumor but independent from free p53 protein serum levels and p53 gene mutations. In contrast to CEA, presence of p53 auto-antibodies is not correlated with UICC stage. p53 auto-antibodies seem not to be predictive in poor outcome and irrelevant in colorectal cancer diagnosis. No significant financial relationships to disclose.

Functional localization and competition between the androgen receptor and T-cell factor for nuclear beta-catenin: a means for inhibition of the Tcf signaling axis.

Recent reports suggest that the beta-catenin-T-cell factor (Tcf) (BCT) signaling pathway is important in the progression of prostate cancer. Evidence suggests that the androgen receptor (AR) can repress BCT-mediated transcription both in prostate cancer and colon cancer cells (Chesire and Isaacs, 2002). In this study, we validate such findings and show that repression of BCT signaling is facilitated by competition between the AR and Tcf. Measurements of the Tcf transcriptional reporter (TOPFLASH) indicated that AR+DHT-mediated repression can inhibit BCT transcription in the presence of WT and exogenous activating beta-catenin (Delta1-130 bp). Transient transfections in SW480 cells (APC(mut/mut)) showed that this mode of repression is functionally independent of APC-mediated beta-catenin ubiquitination. Using a recently developed red flourescent protein (HcRed), we demonstrate novel observations about the nuclear distribution of Tcf. Furthermore, with the use of red (HcRed-AR and HcRed-Tcf) and green fusion proteins (beta-catenin-EGFP), we provide morphological evidence of a reciprocal balance of nuclear beta-catenin-EGFP (BC-EGFP). By cotransfecting in LNCaP prostate tumor cells and using quantitative imaging software, we demonstrated a 62.0% colocalization of HcRed-AR and BC-EGFP in the presence of DHT and 63.3% colocalization of HcRed-Tcf/BC-EGFP in the absence of DHT. Costaining for activated RNA Pol II (phosphoserine 2) and HcRed-Tcf suggested that Tcf foci contain transcriptional 'hotspots' validating that these sites have the capacity for transcriptional activity. Given this apparent androgen-dependent competition for nuclear BC-EGFP, we chose to assess our hypothesis by in vivo and in vitro binding assays. SW480 cells transiently transfected with an AR expression construct, treated with DHT and immunoprecipitated for Tcf showed less associated beta-catenin when compared to Tcf precipitates from untreated cells. Furthermore, by treating cells with DHT+Casodex, we were able to abrogate the androgen-sensitive AR/beta-catenin interaction, in addition to relieving transcriptional repression of the TOPFLASH reporter. In vitro binding assays, with increasing amounts of AR(S35), resulted in decreased Tcf(S35) association with immunoprecipitated recombinant beta-catenin-HIS. These data suggest that in steady-state conditions, AR has the ability to compete out Tcf binding for beta-catenin. Finally, using SW480 cells, we show that AR-mediated repression of the BCT pathway has implications for cell cycle progression and in vitro growth. Using FACs analysis, we observed a 26.1% increase in accumulation of cells in the G1 phase of the cell cycle, while in vitro growth assays showed a 35% reduction in viable cells transfected with AR+DHT treatment. Together, our data strongly suggest that a reciprocal balance of nuclear beta-catenin facilitates AR-mediated repression of BCT-driven transcription and cell growth.

Leukocyte adherence contributes to disruption of the blood–brain barrier during activation of mast cells

The goal of this study was to examine the role of leukocytes in disruption of the blood–brain barrier during activation of mast cells using compound 48/80. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood–brain barrier (clearance of fluorescent-labeled dextran; molecular weight 10 000 daltons; FITC-dextran-10 K) was determined while suffusing with vehicle or compound 48/80 (10 or 25 μg/ml). During superfusion with vehicle (saline), clearance of FITC-dextran-10 K from pial vessels was modest and remained relatively constant during the experimental period (0.52±0.05 ml/s×10 −6 at 80 min). In addition, diameter of pial arterioles remained constant (32±5 μm) while suffusing with vehicle. In contrast, topical application of compound 48/80 produced marked disruption of the blood–brain barrier to FITC-dextran-10 K. For example, suffusion with compound 48/80 (25 μg/ml) increased clearance of FITC-dextran-10 K about 4-fold to 2.26±0.25 ml/s×10 −6 at 80 min. In addition, superfusion with compound 48/80 (25 μg/ml) constricted pial arterioles by 26±9% at 80 min. To determine a potential role for leukocyte adhesion to endothelium in disruption of the blood–brain barrier during suffusion with compound 48/80, we examined permeability during suffusion with compound 48/80 (25 μg/ml) in the presence of WT.3 (2 mg/kg i.v.), a monoclonal antibody directed against the functional epitope of the leukocyte adhesive glycoprotein (CD18; LFA-1β). We found that infusion of WT.3 markedly attenuated disruption of the blood–brain barrier to FITC-dextran-10 K in response to compound 48/80. The clearance of FITC-dextran-10 K during superfusion with compound 48/80 in the presence of WT.3 was 1.29±0.14 ml/s×10 −6 at 80 min ( P<0.05). Thus, the findings of the present study suggest that application of compound 48/80, to degranulate mast cells, activates the adhesion of leukocytes to cerebral venular endothelium which contributes to disruption of the blood–brain barrier.

While E1A Can Facilitate Epithelial Cell Transformation by Several Dominant Oncogenes, the C-Terminus Seems Only to Regulate rac and cdc42 Function, but in Both Epithelial and Fibroblastic Cells

Epithelial and fibroblast cells were differentially susceptible to transformation by oncogenic src, ras, mos, raf, rac, and cdc42 and the influence of adenovirus E1A. In contrast to NIH 3T3 cells, which are easily transformed by all the oncogenes tested, epithelial cells were more resistant to transformation by the same oncogenes. Transformation efficiency of both primary and immortal epithelial cells by E1B, V12ras, v-src, v-raf, and v-mos was increased by cotransfection of E1A 12S, which enables these cells to overcome the M1/M2 mortality blocks, which are not present in NIH 3T3 cells. NIH 3T3 cell transformation by these oncogenes was not altered by E1A. Although V12cdc42 or V12rac1 alone could produce foci on NIH 3T3 cells, morphological conversion was observed only in the presence of a hypertransforming E1A mutant and not WT E1A. Epithelial cells were not transformed by V12cdc42 or V12rac1, even in the presence of WT or mutant E1A, but could be transformed by coexpression of mos/raf and rac/cdc42, and the resultant phenotype was affected by the E1A C-terminus. Hypertransformation, which has previously been reported with ras and E1A C-terminal mutants, turns out to be due to a synergy with rac/cdc42, but not ERK/MAPK or PI3K ras effectors. Like V12rac, expression of the E1A hypertransforming mutant resulted in the upregulation of vinculin and VASP, concomitant with the altered organization of the actin cytoskeleton in these cells. The results show that in addition to requiring abrogation of M1/M2 mortality blocks, primary epithelial cells require activation of the ERK MAPK cascade and rearrangement of the actin CSK to achieve transformation. In addition, the E1A C-terminus regulates rac/cdc42 function in both epithelial and fibroblast cells to affect the extent of transformation progression.