Rs2686344 and serum squamous cell carcinoma antigen could predict clinical efficacy of neoadjuvant chemotherapy for cervical cancer.

OBJECTIVE: To evaluate the predictive value of a single nucleotide polymorphism (SNP) rs2686344 and squamous cell carcinoma antigen (SCCAg) levels in the clinical efficacy of neoadjuvant chemotherapy (NACT) for cervical cancer. METHODS: A total of 92 patients with stage IB2-IIIB carcinoma of the uterine cervix who received NACT treatment were enrolled. The relationship between the genotypes of SNP rs2686344 which is located on CAMKK2 on chromosome 12, SCCAg levels and the response to NACT was analyzed. The relationship between the SNP rs2686344 genotypes, SCCAg levels, the response to NACT and the five-year survival rate was evaluated. RESULTS: The effective group accounted for 84.85% in patients with low level (≤3.5 ng/mL) of post-treatment SCCAg (post-SCCAg), while the ineffective group accounted for 15.15%. The post-SCCAg levels and the genotypes of rs2686344 were significantly correlated with NACT response (P = 0.003, and P = 0.006). In patients with CC or CT genotype of SNP rs2686344, effective group accounted for 81.18%, while ineffective group accounted for 18.82%; For patients with TT genotype, effective response group accounted for 28.57%, ineffective group accounted for 71.43%. Post-SCCAg level >3.5 ng/mL and TT genotype of SNP rs2686344 showed as independent risk factors for NACT response in the multivariate analysis (P = 0.002, and P = 0.048). There was no significant difference in 5-year overall survival and 5-year disease-free survival between patients with different levels of post-SCCAg, or among different rs2686344 genotypes. CONCLUSION: The high level of post-SCCAg (>3.5 ng/mL) and TT genotype of rs2686344 may suggest a higher risk of poor response to NACT.

The role of vaspin in porcine corpus luteum.

Vaspin, visceral adipose tissue-derived serine protease inhibitor, plays important roles in inflammation, obesity, and glucose metabolism. Our recent research has shown the expression and role of vaspin in the function of ovarian follicles. However, whether vaspin regulates steroidogenesis and luteolysis in the corpus luteum (CL) is still unknown. The aim of this study was first to determine the expression of vaspin and its receptor GRP78 in porcine CL at the early, middle, and late stages of the luteal phase. Next, we investigated the hormonal regulation of vaspin levels in luteal cells in response to luteinizing hormone (LH), progesterone (P4), and prostaglandin PGE2 and PGF2α. Finally, we determined vaspin's direct impact on luteal cells steroidogenesis, luteolysis and kinases phosphorylation. Our results are the first to show higher vaspin/GRP78 expression in middle and late vs early stages; immunohistochemistry showed cytoplasmic vaspin/GRP78 localization in small and large luteal cells. In vitro, we found that LH, P4, PGE2, and PGF2α significantly decreased vaspin levels. Furthermore, vaspin stimulated steroidogenesis by the activation of the GRP78 receptor and protein kinase A (PKA). Also, vaspin increased the ratio of luteotropic PGE2 to luteolytic PGF2α secretion via GRP78 and mitogen-activated kinase (MAP3/1). Moreover, vaspin, in a dose-dependent manner, decreased GRP78 expression, while it, in a time-dependent manner, increased kinases PKA and MAPK3/1 phosphorylation. Taken together, we found that vaspin/GRP78 expression depends on the luteal phase stage and vaspin affects luteal cells endocrinology, indicating that vaspin is a new regulator of luteal cells steroidogenesis and CL formation.

Pigment epithelium-derived factor from ARPE19 promotes proliferation and inhibits apoptosis of human umbilical mesenchymal stem cells in serum-free medium.

Clinical expansion of mesenchymal stem cells (MSCs) is hampered by the lack of knowledge regarding how to prevent MSC apoptosis and promote their proliferation in serum-free medium. Our in vitro studies demonstrated that human umbilical cord MSCs (HUCMSCs) underwent apoptosis in the serum-free medium. When HUCMSCs were co-cultured with retinal pigment epithelial cells (ARPE19), however, HUCMSCs exhibited normal growth and morphology in serum-free medium. Their colony formation was promoted by the conditioned medium (CM) of ARPE19 cells on Matrigel. Proteomics analysis showed that pigment epithelium-derived factor (PEDF) was one of the most abundant extracellular proteins in the ARPE19 CM, whereas enzyme-linked immunosorbent assay confirmed that large amounts of PEDF was secreted from ARPE19 cells. Adding anti-PEDF-blocking antibodies to the co-culture of HUCMSCs with ARPE19 cells increased apoptosis of HUCMSCs. Conversely, treatment with PEDF significantly reduced apoptosis and increased proliferation of HUCMSCs in serum-free medium. PEDF was further demonstrated to exert this anti-apoptotic effect by inhibiting P53 expression to suppress caspase activation. In vivo studies demonstrated that co-injection of HUCMSCs with ARPE19 cells in immunocompromised NOD-SCID mice also increased survival and decreased apoptosis of HUCMSCs. PEDF also showed no negative effect on the mesoderm differentiation capability of HUCMSCs. In conclusion, this study is the first to demonstrate that PEDF promotes HUCMSC proliferation and protects them from apoptosis by reducing p53 expression in the serum-free medium. This study provides crucial information for clinical-scale expansion of HUCMSCs.

Changes of inflammatory factors after intravitreal triamcinolone acetonide for macular edema with central retinal vein occlusion.

PURPOSE: To investigate the effect of intravitreal triamcinolone acetonide (TA) on aqueous humor levels of vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule 1 (sICAM-1), and pigment epithelium-derived factor (PEDF) in patients with central retinal vein occlusion (CRVO) and macular edema. METHODS: We measured VEGF, sICAM-1, and PEDF levels in aqueous humor samples from 2 eyes of 2 CRVO patients during injection of TA. RESULTS: In both patients, the VEGF and sICAM-1 levels in aqueous humor samples obtained during initial injection of TA were higher than at the time of reinjection. Conversely, the initial PEDF levels were lower than those at reinjection. CONCLUSIONS: Aqueous humor levels of VEGF and sICAM-1 were decreased by TA treatment in 2 CRVO patients, while PEDF was increased. Intravitreal TA could be an option for CRVO patients with a low PEDF level and/or moderate VEGF and sICAM-1 levels.

Influence of lactic acid on differential expression of vascular endothelial growth factor and pigment epithelium-derived factor in explants of rat retina.

PURPOSE: To investigate the influence of lactate on expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in rat retina. METHODS: Retinal explants from neonatal Sprague Dawley rats were incubated with media containing 10, 20, or 30 mM of lactic acid. The 10 mM group was used as a control. At 24 h after incubation, retinas were sectioned for light microscopy, and expressions of VEGF and PEDF measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: The architecture of cultured retinas appeared to be intact. Compared with control, both RT-PCR and Western blot analysis showed that 30 mM of lactic acid significantly increased the levels of VEGF, but not PEDF. CONCLUSIONS: Stimulation of production of retinal VEGF by lactate is dependent on the concentration of lactate. Lactate has no effect on the expression of PEDF in rat retinal explants.

Isoliquiritigenin from licorice root suppressed neovascularisation in experimental ocular angiogenesis models.

AIM: To explore the antiangiogenic property of isoliquiritigenin (ISL) on in vivo and in vitro models. DESIGN: Laboratory investigation. METHODS: The effect of ISL on angiogenesis development was investigated using ex ovo chick chorioallantoic membrane model. Its effect on pathological angiogenesis was examined by (1) silver nitrate cauterisation-induced corneal neovascularisation in BALB/c mice, followed by topical ISL (0.2-50 µM) and CD31 immunofluorescence of corneal blood vessels; (2) argon laser photocoagulation-induced choroidal neovascularisation in C57BL/6 mice, followed by intravitreal ISL (10-200 µM) and fundus fluorescein angiography and immunofluorescence with Griffonia simplicifolia isolectin-B4 (GSA I-B4); and (3) oxygen-induced retinopathy in C57BL/6J mice pups, followed by intravitreal ISL (1-100 µM) and GSA I-B4 immunofluorescence. The vascular area was quantified and analysed by one-way analysis of variance and Student t test. Expression of vascular endothelial growth factor (VEGF) and pigment-epithelium-derived factor in human umbilical vein endothelial cells was analysed by western blotting. RESULTS: Ex ovo chick chorioallantoic membrane assay showed that ISL dose-dependently suppressed VEGF-induced vessel growth. In vivo experiments illustrated that topical ISL alleviated corneal neovascularisation (IC(50)=7.14 µM, day 7) and intravitreal ISL reduced vessel leakage and GSA I-B4-positive vascular area in choroidal and retinal neovascularisation. ISL was found to dose-dependently suppress VEGF and induce pigment epithelium derived factor expression in cultured endothelial cells. CONCLUSION: Using various experimental models of ocular neovascularisation, the authors have demonstrated that ISL from licorice extract has an antiangiogenic effect. The authors' findings suggest that ISL may be a potential antiangiogenic molecule in the development of therapy for neovascularisation diseases.

Effects of acrolein, a natural occurring aldehyde, on the anticoagulant serpin antithrombin.

We studied the effect of acrolein, an alpha,beta-unsaturated aldehyde that causes adduct-modification of lysine, cysteine, and histidine residues, on antithrombin, a key anticoagulant serpin. Intrinsic fluorescence, functionality (anti-FXa and anti-IIa activity), heparin affinity and conformational features of plasma and purified antithrombin were evaluated. In vivo experiments were carried out in mice. Intrinsic fluorescence showed a two-step conformational change. Acrolein, even at low dose, impaired the anticoagulant function of purified antithrombin by affecting its heparin affinity. However, higher concentrations of acrolein and long incubations are required to cause mild functional effects on plasma antithrombin and mice.

Differential expression of anti-angiogenic factors and guidance genes in the developing macula.

PURPOSE: The primate retina contains a specialized, cone-rich macula, which mediates high acuity and color vision. The spatial resolution provided by the neural retina at the macula is optimized by stereotyped retinal blood vessel and ganglion cell axon patterning, which radiate away from the macula and reduce shadowing of macular photoreceptors. However, the genes that mediate these specializations, and the reasons for the vulnerability of the macula to degenerative disease, remain obscure. The aim of this study was to identify novel genes that may influence retinal vascular patterning and definition of the foveal avascular area. METHODS: We used RNA from human fetal retinas at 19-20 weeks of gestation (WG; n=4) to measure differential gene expression in the macula, a region nasal to disc (nasal) and in the surrounding retina (surround) by hybridization to 12 GeneChip microarrays (HG-U133 Plus 2.0). The raw data was subjected to quality control assessment and preprocessing, using GC-RMA. We then used ANOVA analysis (Partek) Genomic Suite 6.3) and clustering (DAVID website) to identify the most highly represented genes clustered according to "biological process." The neural retina is fully differentiated at the macula at 19-20 WG, while neuronal progenitor cells are present throughout the rest of the retina. We therefore excluded genes associated with the cell cycle, and markers of differentiated neurons, from further analyses. Significantly regulated genes (p<0.01) were then identified in a second round of clustering according to molecular/reaction (KEGG) pathway. Genes of interest were verified by quantitative PCR (QRT-PCR), and 2 genes were localized by in situ hybridization. RESULTS: We generated two lists of differentially regulated genes: "macula versus surround" and "macula versus nasal." KEGG pathway clustering of the filtered gene lists identified 25 axon guidance-related genes that are differentially regulated in the macula. Furthermore, we found significant upregulation of three anti-angiogenic factors in the macula: pigment epithelium derived factor (PEDF), natriuretic peptide precurusor B (NPPB), and collagen type IValpha2. Differential expression of several members of the ephrin and semaphorin axon guidance gene families, PEDF, and NPPB was verified by QRT-PCR. Localization of PEDF and Eph-A6 mRNAs in sections of macaque retina shows expression of both genes concentrates in the ganglion cell layer (GCL) at the developing fovea, consistent with an involvement in definition of the foveal avascular area. CONCLUSIONS: Because the axons of macular ganglion cells exit the retina from around 8 WG, we suggest that the axon guidance genes highly expressed at the macula at 19-20 WG are also involved in vascular patterning, along with PEDF and NPPB. Localization of both PEDF and Eph-A6 mRNAs to the GCL of the developing fovea supports this idea. It is possible that specialization of the macular vessels, including definition of the foveal avascular area, is mediated by processes that piggyback on axon guidance mechanisms in effect earlier in development. These findings may be useful to understand the vulnerability of the macula to degeneration and to develop new therapeutic strategies to inhibit neovascularization.

Vitamin D reduces the expression of collagen and key profibrotic factors by inducing an antifibrotic phenotype in mesenchymal multipotent cells.

Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5'-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0.1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.

A phase II trial of 17-allylamino-17-demethoxygeldanamycin in patients with hormone-refractory metastatic prostate cancer.

PURPOSE: 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic with antiproliferative activity in several mouse xenograft models, including prostate cancer models. A two-stage phase II study was conducted to assess the activity and toxicity profile of 17-AAG administered to patients with metastatic, hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: Patients with at least one prior systemic therapy and a rising prostate-specific antigen (PSA) were eligible. Patients received 17-AAG at a dose of 300 mg/m2 i.v. weekly for 3 of 4 weeks. The primary objective was to assess the PSA response. Secondary objectives were to determine overall survival, to assess toxicity, and to measure interleukin-6, interleukin-8, and maspin levels and quality of life. RESULTS: Fifteen eligible patients were enrolled. The median age was 68 years and the median PSA was 261 ng/mL. Patients received 17-AAG for a median number of two cycles. Severe adverse events included grade 3 fatigue (four patients), grade 3 lymphopenia (two patients), and grade 3 back pain (two patients). The median PSA progression-free survival was 1.8 months (95% confidence interval, 1.3-3.4 months). The 6-month overall survival was 71% (95% confidence interval, 52-100%). CONCLUSIONS: 17-AAG did not show any activity with regard to PSA response. Due to insufficient PSA response, enrollment was stopped at the end of first stage per study design. The most significant severe toxicity was grade 3 fatigue. Further evaluation of 17-AAG at a dose of 300 mg/m2 i.v. weekly as a single agent in patients with metastatic, hormone-refractory prostate cancer who received at least one prior systemic therapy is not warranted.

Inhibition of plasminogen activator inhibitor-1: its mechanism and effectiveness on coagulation and fibrosis.

OBJECTIVE: Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. METHODS AND RESULTS: We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A beta-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride-induced testicular artery thrombosis. It also prevents the fibrotic process initiated by bleomycin in mouse lung. CONCLUSIONS: The present study demonstrates beneficial in vitro and in vivo effects of novel PAI-1 inhibitors. Our methodology proves to be a useful tool to obtain effective inhibitors of serpin activity.

Tumor transcriptome reveals the predictive and prognostic impact of lysosomal protease inhibitors in non-small-cell lung cancer.

PURPOSE: Insight into clinical response to platinum-based chemotherapy (PBC) in non-small-cell lung cancer (NSCLC). METHODS: Matched tumor and nontumor lung tissues from PBC-treated NSCLC patients (four nonresponders and four responders) and tumor tissue from an independent test set (four nonresponders and four responders), were profiled using microarrays. Lysosomal protease inhibitors SerpinB3 and cystatin C were highly correlated with clinical response and were further evaluated by immunohistochemistry in PBC-treated patients (36 prechemotherapy and 13 postchemotherapy). Investigation of the pathogenic and prognostic significance of SerpinB3 was performed in 251 primary tumors, with 64 regional lymph node pairs, from chemotherapy-naïve NSCLC patients using immunohistochemistry. RESULTS: Bioinformatic analyses of gene expression in the training set identified a gene set (n = 17) that separated all patients in the training and test sets (n = 16) according to response in hierarchical clustering. Transcriptome profiling revealed that SerpinB3 mRNA was highly correlated with degree of response (r = -0.978; P < .0001) and was a clear outlier (nonresponders:responders > 50-fold). SerpinB3 protein expression was correlated with clinical response in PBC-treated NSCLC patients (P = .045). Expression of SerpinB3 and cystatin C, relative to the target, protease cathepsin B, was independently predictive of response (odds ratio, 17.8; 95% CI, 2.0 to 162.4; P = .01), with an accuracy of 72%. High SerpinB3 expression levels, invariably associated with chemoresistance, had contrasting prognostic impact in untreated squamous cell carcinomas (hazard ratio [HR], 0.43; 95% CI, 0.18 to 0.93) or adenocarcinomas (HR, 2.09; 95% CI, 1.03 to 4.72). CONCLUSION: This provides the first comprehensive molecular characterization of clinical responsiveness to PBC in NSCLC and reveals the predictive and prognostic impact of two lysosomal protease inhibitors, potentially representing novel targets for NSCLC therapeutics.

Basal cell adhesion molecule is inversely associated with apoptosis, but plays a limited role for protection against apoptotic stimuli.

Basal cell adhesion molecule (B-CAM) is strongly upregulated in epithelial skin cancer cells in vivo and in vitro. We have tested here whether B-CAM is (1) inversely associated with or (2) functionally involved in apoptosis. Towards this end, B-CAM expression was assessed in HaCaT transfectants overexpressing murine Bcl-2 and untransfected HaCaT cells exposed to various proapoptotic stimuli. In another series of experiments, we overexpressed B-CAM in HaCaT cells and different fibroblast lines, and stimulated various apoptotic pathways in the transfectants and control cells. In addition, apoptosis was assessed after an antibody-mediated B-CAM blockade. We could demonstrate that expression of B-CAM is inversely associated with the susceptibility of cells to apoptosis. However, overexpression or antibody- mediated inhibition of B-CAM had only limited functional effects on cellular apoptosis.

Pigment epithelium-derived factor in idiopathic pulmonary fibrosis: a role in aberrant angiogenesis.

Pigment epithelium-derived factor (PEDF) is a 50-kD protein with angiostatic and neurotrophic activities that regulates vascular development within the eye. PEDF expression was increased in the lungs of patients with idiopathic pulmonary fibrosis (IPF) based on microarray analyses. Angiogenesis has been implicated in the pathogenesis of fibrotic lung diseases, we therefore hypothesized that regional abnormalities in vascularization occur in IPF as a result of an imbalance between PEDF and vascular endothelial growth factor. We demonstrated that vascular density is regionally decreased in IPF within the fibroblastic foci, and that within these areas PEDF was increased, whereas vascular endothelial growth factor was decreased. PEDF colocalized with the fibrogenic cytokine, transforming growth factor (TGF)-beta 1, particularly within the fibrotic interstitium and the fibroblastic focus, and prominently within the epithelium directly overlying the fibroblastic focus. This suggested that TGF-beta 1 might regulate PEDF expression. Using 3T3-L1 fibroblasts and human lung fibroblasts, we showed that PEDF was indeed a TGF-beta 1 target gene. Collectively, our findings implicate PEDF as a regulator of pulmonary angiogenesis and an important mediator in IPF.

Disruption of cell-type-specific methylation at the Maspin gene promoter is frequently involved in undifferentiated thyroid cancers.

Cancer-associated DNA hypomethylation is as prevalent as cancer-linked hypermethylation, but the biological significance of DNA hypomethylation in carcinogenesis is less understood. The expression of Maspin (mammary serpin) in differentiated normal cells is regulated by epigenetic modifications in a cell-type-specific manner. Paradoxical Maspin expression due to epigenetic modification has been addressed in several cancer cell types. To elucidate the role of the Maspin gene in thyroid cancer, we studied methylation status in the promoter region and its expression in six human undifferentiated thyroid cancer cell lines and in specimens from 92 primary thyroid tumors, consisting of six follicular adenomas, 56 well-differentiated thyroid cancers (WDTCs), 17 poorly differentiated thyroid cancers (PDTCs) and 13 undifferentiated thyroid cancers (UDTCs). Three of the six cell lines overexpressed Maspin mRNA and its protein product, but the remaining three did not. The methylation status at the promoter region was inversely correlated with Maspin expression. In Maspin-negative cell lines, Maspin expression was induced by treatment with 5-aza-2'-deoxycytidine, a DNA demethylating agent. Immunoreactivity for Maspin protein was frequently detected in UDTCs (8/13, 62%) and PDTCs (7/17, 41%). Immunoreactivity for Maspin was diffusely positive in UDTCs, and was restricted to dedifferentiated components of the tumor in PDTCs. Positive immunoreactivity was infrequent in WDTCs (1/56, 2%), and all follicular adenomas and normal thyroid glands were completely negative. Their methylation status evaluated by the methylation-specific PCR method showed a good inverse correlation with their immunoreactivity in surgically resected specimens. Our data suggest that overexpression of Maspin by DNA hypomethylation is closely associated with morphological dedifferentiation in thyroid cancers.

A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1).

Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

Tamoxifen enhances myoepithelial cell suppression of human breast carcinoma progression in vitro by two different effector mechanisms.

Our previous studies have indicated that myoepithelial cells surrounding ductal and acinar epithelium of glandular organs, such as the breast, exert multiple paracrine suppressive effects on incipient and developing cancers that arise from this epithelium. Myoepithelial cells and derived cell lines (HMS 1-6) exert these effects through the secretion of a number of different effector molecules that exert anti-invasive, anti-proliferative, and anti-angiogenic activities. Since previous basic and clinical studies have examined the role of estrogen agonists and antagonists on human breast cancer cells and because issues of hormone replacement therapy (HRT) and tamoxifen chemoprevention are such timely issues in breast cancer, we wondered whether or not hormonal manipulations might affect myoepithelial cells in vitro as far as their paracrine suppressive activities on breast cancer were concerned. The present in vitro study demonstrates that treatment of myoepithelial cells with tamoxifen but not 17beta-estradiol increases both maspin secretion and invasion-blocking ability. Furthermore tamoxifen but not 17beta-estradiol increases inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by myoepithelial cells when they are co-cultured with conditioned media from or breast carcinoma cells directly. This increased myoepithelial NO exerts both autocrine and paracrine antiproliferative effects which can be blocked by inhibition of iNOS. 17beta-Estradiol, however, competes with all of these suppressive effects of tamoxifen suggesting that the mechanism of tamoxifen action is estrogen receptor mediated. Myoepithelial cells lack ER-alpha but express ER-beta. Tamoxifen, but not 17beta-estradiol, increases AP-1 CAT but not ERE-CAT activity. Again, 17beta-estradiol competes with the transcription-activating effects of tamoxifen. These experiments collectively suggest that the actions of tamoxifen on the increased secretion of maspin and increased production of NO by myoepithelial cells are mediated through ER-beta and the transcription-activation of an ER-dependent AP-1 response element.

Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3.

To elucidate structure-function relationships of stromelysin-3, a putative matrix metalloproteinase originally identified at the tumor-stromal cell interface in breast carcinomas, the human cDNA was expressed in mammalian cells, and its products were isolated and characterized. In stably transfected cells, stromelysin-3 was recovered as a complex mixture of species ranging in size from approximately 20 to 65 kDa. Among these products, a major 45-kDa species with an N terminus of Phe98 and an intact C-terminal domain was identified as a true endopeptidase on the basis of its ability to cleave the bait region of alpha 2-macroglobulin between Phe684 and Tyr685, a site identical to that recognized by stromelysin-1. However, unlike stromelysin-1 or other members of the matrix metalloproteinase family, the mature form of stromelysin-3 was unable to hydrolyze a range of extracellular matrix molecules associated with either the basement membrane or interstitium. To probe for alternate substrates among tumor cell-derived products, purified stromelysin-3 was incubated with [35S]methionine-labeled medium conditioned by the breast cancer cell line, MCF-7. Under these conditions, a single, tumor cell-derived protein was hydrolyzed as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Following anion-exchange chromatography and preparative gel electrophoresis, the stromelysin-3 substrate was identified by N-terminal sequencing as the serine proteinase inhibitor, alpha 1-proteinase inhibitor. Further studies demonstrated that stromelysin-3 rapidly destroyed the antiproteolytic function of alpha 1-proteinase inhibitor by cleaving the antiproteinase at a distinct site between Ala350 and Met351 within the reactive-site loop. Together, these data not only demonstrate that human stromelysin-3 acts as a powerful endopeptidase with a restricted substrate specificity distinct from all other matrix metalloproteinases, but also serve to identify serine proteinase inhibitors as potential physiologic targets at sites of extracellular matrix remodeling.