[Show abstract][Hide abstract] ABSTRACT: Endometriosis is characterized by the presence of functional endometrial tissue outside of the uterine cavity. It affects 1 in 10 women of reproductive age. This chronic condition commonly leads to consequences such as pelvic pain, dysmenorrhea, infertility and an elevated risk of epithelial ovarian cancer. Despite the prevalence of endometriosis and its impact on women's lives, there are relatively few in vitro and in vivo models available for studying the complex disease biology, pathophysiology, and for use in the preclinical development of novel therapies. The goal of this study was to develop a novel three-dimensional (3D) cell culture model of ovarian endometriosis and to test whether it is more reflective of endometriosis biology than traditional two dimensional (2D) monolayer cultures.
A novel ovarian endometriosis epithelial cell line (EEC16) was isolated from a 34-year old female with severe endometriosis. After characterization of cells using in vitro assays, western blotting and RNA-sequencing, this cell line and a second, already well characterized endometriosis cell line, EEC12Z, were established as in vitro 3D spheroid models. We compared biological features of 3D spheroids to 2D cultures and human endometriosis lesions using immunohistochemistry and real-time semi-quantitative PCR.
In comparison to normal ovarian epithelial cells, EEC16 displayed features of neoplastic transformation in in vitro assays. When cultured in 3D, EEC16 and EEC12Z showed differential expression of endometriosis-associated genes compared to 2D monolayer cultures, and more closely mimicked the molecular and histological features of human endometriosis lesions.
To our knowledge, this represents the first report of an in vitro spheroid model of endometriosis. 3D endometriosis models represent valuable experimental tools for studying EEC biology and the development of novel therapeutic approaches.
Journal of Ovarian Research 02/2014; 7(1):17. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model. DESIGN: Experimental laboratory study. SETTING: University medical centers. PATIENT(S): Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. INTERVENTION(S): The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti-miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. MAIN OUTCOME MEASURE(S): Predicted target expression was measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. RESULT(S): In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre-miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. CONCLUSION(S): miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.
Fertility and sterility 01/2013; · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endometriosis is a chronic gynecological disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors EP2 and EP4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of EP2 and EP4-mediated PGE2 signaling: (i) decreases expression and/or activity specific integrin receptor subunits beta1 and beta3 but not beta5, alpha1, alpha2, alpha5, and alphav subunits; (ii) decreases integrin signaling components focal adhesion kinase (FAK) and talin proteins; (iii) inhibits interactions between beta1/beta3 subunits, FAK and talin and EP2/EP4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and (iv) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.
Biology of Reproduction 12/2012; · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To study the function of syndecan-1 (SDC1) and its potential regulator miR-10b in endometriosis. DESIGN: Experimental laboratory study. SETTING: University medical center. PATIENT(S): Not applicable. INTERVENTION(S): The human endometriotic cell line 12Z was transiently transfected with SDC1 small interfering RNA or miR-10b precursors and investigated for changes in cell behavior and gene expression. 12Z and primary eutopic endometrial stroma cells of two American Society for Reproductive Medicine class III endometriosis patients were transfected with miR-10b precursors to investigate posttranscriptional regulation of SDC1. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction, Western blotting, flow cytometry, 3' untranslated region luciferase assays, and zymography were used to measure miR-10b-dependent targeting of SDC1 and SDC1-dependent expression changes of proteases and interleukin-6. Altered cell behavior was monitored by Matrigel invasion assays, cell viability assays, and mitogen-activated protein kinase activation blots. RESULT(S): Knockdown of SDC1 inhibited Matrigel invasiveness by >60% but did not affect cell viability. Interleukin-6 secretion, matrix metalloproteinase-9 expression, and matrix metalloproteinase-2 activity were reduced, whereas plasminogen activator inhibitor-1 protein expression was up-regulated. miR-10b overexpression significantly down-regulated SDC1, reduced Matrigel invasiveness by 20% and cell viability by 14%, and decreased mitogen-activated protein kinase activation in response to hepatocyte growth factor. CONCLUSION(S): Syndecan-1, a target of miR-10b, inhibits epithelial endometriotic cell invasiveness through down-regulation of metalloproteinase activity and interleukin-6.
Fertility and sterility 11/2012; · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cathepsin B has been shown to be important in angiogenesis; therefore, understanding its regulation in endothelial cells should provide fundamental information that will aid in the development of new treatment options. Peroxisome proliferator-activated receptors (PPARs) have been shown to have anti-inflammatory, anti-angiogenic and anti-tumorigenic properties. We explored the influence of a PPARα agonist on cathepsin B expression in human endothelial cells. The PPARα agonist, Wy14643, was found to inhibit cathepsin B protein expression. Further studies demonstrated the Wy14643-dependent but PPARα-independent suppression of cathepsin B. This has been previously described for other PPAR agonists. Wy14643 suppressed the accumulation of cathepsin B mRNA, which was accompanied by the selective suppression of a 5'-alternative splice variant. Consistent with these results, luciferase promoter assays and electrophoretic mobility shift analysis demonstrated that the suppression was facilitated by reduced binding of the transcription factors USF1/2 to an E-box within the cathepsin B promoter. Additionally, Wy14643 treatment resulted in a reduction in cathepsin B half-life, suggesting a posttranslational regulatory mechanism. Overall, our results suggest that the PPARα-dependent anti-angiogenic action of Wy14643 seems to be mediated, in part, by Wy14643-dependent but PPARα-independent regulation of cathepsin B expression.
[Show abstract][Hide abstract] ABSTRACT: Peroxisome proliferator-activated receptor (PPAR) delta agonists are known to have distinct anti-inflammatory and antitumor effects; though, the knowledge regarding their mode of action has thus far been limited. Different cathepsins have been shown to be upregulated in a broad range of pathological events, such as rheumatoid arthritis, psoriasis, atherosclerosis and diverse tumor entities, for example melanoma. Recent work demonstrated that cathepsin B in particular is an important pro-angiogenic protease in various pathological conditions. We therefore analysed whether cathepsins are a valid target for PPARδ agonists. This study reveals an inhibitory effect of two commonly used PPARδ agonists, GW501516 and L-165,041, on the protein expression and enzyme activity of cathepsin B in human endothelial cells. In contrast, no inhibitory effects were observed on cathepsin L and cathepsin D protein expression after treatment with PPARδ agonists. Furthermore, the results substantiate that PPARδ activators mediate their inhibitory action in a PPARδ-dependent manner and that the underlying regulatory mechanism is not based on a transcriptional but rather on a posttranslational mode of action, via the reduction in the cathepsin B protein half-life. Mechanisms conveying the suppressive effect by 5'-alternative splicing, a 3'-UTR-dependent way or by miRNA could be excluded. The data of this study explore cathepsin B as a new valid target for PPARδ agonists in endothelial cells. The results bolster other studies demonstrating PPARδ agonists as anti-inflammatory and anticarcinogenic agents and thus might have the potential to help to develop new pharmaceutical drugs.
[Show abstract][Hide abstract] ABSTRACT: Targeting of proteins to the endoplasmic reticulum (ER) usually requires N-terminal signal peptides (SP) of approximately 22 amino acids in length. However, a substantial number of proteins contain exceptionally long SPs of 40 amino acids and more, an example being protein shrew-1/AJAP1. Using shrew-1's SP as example, the NtraC model has been developed by dissecting long SPs into two functionally distinct subdomains ("N" and "C") separated by a β-turn rich transition area ("tra"). Further proteins have been identified by computational analysis complying with the NtraC model. Here we used the SPs of two of these proteins, DCBLD2 and RGMa (including three isoforms), to show that the NtraC model applies to a growing group of SPs. We demonstrate that the full-length SPs of RGMa and DCBLD2 are functional and furthermore that the C-domains are sufficient and essential for ER targeting, whereas the N-domains are dispensable. Thus, the N-domains are available for additional functions.
[Show abstract][Hide abstract] ABSTRACT: Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. We recently reported that inhibition of COX-2 decreased migration as well as invasion of human endometriotic epithelial and stromal cells. Results of the present study indicates that selective inhibition of PGE2 receptors EP2 and EP4 suppresses expression and/or activity of MMP1, MMP2, MMP3, MMP7 and MMP9 proteins and increases expression of TIMP1, TIMP2, TIMP3, and TIMP4 proteins and thereby decreases migration and invasion of human immortalized endometriotic epithelial and stromal cells into matrigel. The interactions between EP2/EP4 and MMPs are mediated through Src and β-arrestin 1 protein complex involving MT1-MMP and EMMPRIN in human endometriotic cells. These novel findings provide an important molecular and cellular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy for endometriosis in childbearing-age women.
Molecular and Cellular Endocrinology 01/2011; 332(1-2):306-13. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel therapeutic approaches for endometriosis based on molecular strategies may prove to be useful. Conditionally replicative adenoviruses (CRAds) are designed to exploit key differences between target and normal cells. The wild-type adenovirus (Adwt) promoter can be replaced by tissue-specific promoters, allowing viral replication only in target cells. Viral infectivity can be enhanced by altering Ad tropism via fiber modification. We investigated whether CRAds can be used to target endometriosis and determined the most efficient transcriptional- and transductional-targeting strategy.
An in vitro study was carried out using human endometriotic cell lines, 11Z (epithelial) and 22B (stromal), normal human ovarian surface epithelial cell line (NOSE006) and primary human endometriosis cells. A total of 9 promoters and 12 Ad tropism modifications were screened by means of a luciferase reporter assay. From this screening data, three CRAds (CRAd-S-pK7, CRAd-S-RGD, CRAd-S-F5/3sigma1, all incorporating the survivin promoter but with different fiber modifications) were selected to perform experiments using Adwt and a replication-deficient virus as controls. CRAds were constructed using a plasmid recombination system. Viral-binding capacity, rates of entry and DNA replication were evaluated by quantitative real-time PCR of viral genome copy. Cell-killing effects were determined by crystal violet staining and a cell viability assay for different concentrations of viral particles per cell.
Comparison of promoters demonstrated that the survivin promoter exhibited the highest induction in both endometriotic cell lines. Among the fiber-modified viruses, the polylysine modification (pK7) showed the best infection enhancement. CRAd-S-pK7 was validated as the optimal CRAd to target endometriosis in terms of binding ability, entry kinetics, DNA replication and cell-killing effect. CRAd-S-pK7 also exhibited a high level of DNA replication in primary endometriosis cells.
CRAd-S-pK7 has the best infection and cell-killing effect in the context of endometriosis. It could prove to be a useful novel method to target refractory cases of endometriosis.
Human Reproduction 08/2010; 25(8):2068-83. · 4.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine interactions between prostaglandin (PG) E(2) signaling and proliferation of endometriotic cells to increase our knowledge about PGE(2) signaling in the pathogenesis of endometriosis in humans.
Immortalized human endometriotic epithelial and stromal cells were used as an in vitro model. Effects of inhibition of PGE(2) receptors on proliferation of endometriotic cells and associated cell cycle regulation were determined.
College Veterinary Medicine and Biomedical Sciences, Texas A&M University.
Cell proliferation, cell viability, cell cycle, regulation of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors.
Selective blockade of EP2/EP4 inhibited proliferation of human endometriotic cells by inducing cell cycle arrest at the G(1)-S and G(2)-M checkpoints in epithelial cells and at the G(2)-M checkpoint in stromal cells. This cell cycle arrest during specific checkpoints was associated with distinct regulation of respective cyclins and cyclin-dependent kinases. Inhibition of EP1 did not decrease endometriotic cell proliferation.
For the first time data from the present study provide a direct molecular link between PGE(2) signaling and proliferation of endometriotic cells and suggest that inhibition of EP2/EP4 could be a potential nonestrogen (E) treatment option for endometriosis in women.
Fertility and sterility 03/2010; 93(8):2498-506. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of anti-L1-cell adhesion molecule monoclonal antibodies (anti-L1CAM-mAb) in an endometriosis epithelial cell line Z12 led to a statistically significant decrease in cell proliferation and cell invasion and to an inhibition of the adhesion compared with unspecific IgG-Ab treated and untreated cells. Because it increases the cell invasion and adhesion which consequently aggravates the disease, L1 could possibly promote endometriosis development; thus, further studies should evaluate the possible use of anti-L1-mAb in an animal endometriosis model.
Fertility and sterility 02/2010; 94(3):1102-4. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine whether nuclear factor-kappaB (NF-kappaB) is constitutively and tumor necrosis factor (TNF)-dependently activated in endometriotic cells, whether trichostatin A (TSA) can suppress NF-kappaB activation and suppress TRAF2/6 and TAK1, and whether TSA and caffeic acid phenyl ester can suppress constitutive and H(2)O(2)-stimulated proliferation of endometriotic cells.
Two endometriotic cell lines and an endometrial stromal cell line were used as an in vitro model. Electrophoretic mobility shift analysis was used to determine NF-kappaB activation and possible suppression by TSA. Western blot analysis was used to determine whether TSA suppresses phosphorylation of IkappaBalpha, phosphorylation of p65 in the cytoplasm and nuclear translocation, and the expression of TRAF2/6 and TAK1.
NF-kappaB was constitutively activated in endometriotic cells, but only minimally in endometrial cells. TNFalpha stimulation activated NF-kappaB through induction of IkappaB phosphorylation, but the activation can be suppressed by TSA. TSA also attenuated constitutive and TNF-dependent p65 phosphorylation and nuclear translocation in endometriotic cells. TRAF2, TRAF6 and TAK1 were constitutively activated and were unaffected by TSA treatment.
NF-kappaB activation may play a critical role in the pathogenesis in endometriosis. Targeting NF-kappaB with histone deacetylase inhibitors or other compounds might hold promise as novel therapeutics for endometriosis.
Gynecologic and Obstetric Investigation 01/2010; 70(1):23-33. · 1.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma spheroid cultures are enriched in tumor stem-like cells and therefore may be more representative of the respective primary tumors than conventional monolayer cultures. We exploited the glioma spheroid culture model to find novel tumor-relevant genes.
We carried out array-based comparative genomic hybridization of spheroid cultures derived from 20 glioblastomas. Microarray-based gene expression analysis was applied to determine genes with differential expression compared with normal brain tissue and to nonneoplastic brain spheroids in glioma spheroid cultures. The protein expression levels of three candidates were determined by immunohistochemistry on tissue microarrays and correlated with clinical outcome. Functional analysis of PDPN was done.
Genomic changes in spheroid cultures closely resembled those detected in primary tumors of the corresponding patients. In contrast, genomic changes in serum-grown monolayer cultures established from the same patients did not match well with the respective primary tumors. Microarray-based gene expression analysis of glioblastoma spheroid cultures identified a set of novel candidate genes being upregulated or downregulated relative to normal brain. Quantitative real-time PCR analyses of 8 selected candidate genes in 20 clinical glioblastoma samples validated the microarray findings. Immunohistochemistry on tissue microarrays revealed that expression of AJAP1, EMP3, and PDPN was significantly associated with overall survival of astrocytic glioma patients. Invasive capacity and RhoA activity were decreased in PDPN-silenced spheroids.
We identified a set of novel candidate genes that likely play a role in glioblastoma pathogenesis and implicate AJAP1, EMP3, and PDPN as molecular markers associated with the clinical outcome of glioma patients.
Clinical Cancer Research 11/2009; 15(21):6541-50. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gain- and loss-of-function studies indicate that the adherens junction protein shrew-1 acts as a novel modulator of E-cadherin internalization induced by epithelial growth factor (EGF) or E-cadherin function-blocking antibody during epithelial cell dynamics. Knocking down shrew-1 in MCF-7 carcinoma cells preserves E-cadherin surface levels upon EGF stimulation. Overexpression of shrew-1 leads to preformation of an E-cadherin/EGF receptor (EGFR) HER2/src-kinase/shrew-1 signaling complex and accelerated E-cadherin internalization. Shrew-1 is not sufficient to stimulate E-cadherin internalization, but facilitates the actions of EGFR and thus may promote malignant progression in breast cancer cells with constitutive EGFR stimulation by reducing surface E-cadherin expression.
Molecular biology of the cell 07/2009; 20(15):3598-607. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endometriosis is a benign chronic gynecological disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, and the reproductive health of women and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen-targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration, and invasion of human endometriotic epithelial and stromal cells, which was due to decreased prostaglandin E(2) (PGE(2)) production. In this study, we determined mechanisms through which PGE(2) promoted survival of human endometriotic cells. Results of the present study indicate that 1) PGE(2) promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, nuclear factor-kappaB, and beta-catenin signaling pathways; 2) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome c, and thus activates caspase-3/poly (ADP-ribose) polymerase-mediated intrinsic apoptotic pathways; and 3) these PGE(2) signaling components are more abundantly expressed in ectopic endometriosis tissues compared with eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including nonestrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.
[Show abstract][Hide abstract] ABSTRACT: Nonresectable ependymomas are associated with poor prognosis despite intensive radiochemotherapy and radiation. The molecular pathogenesis of ependymoma initiation and progression is largely unknown. We here present a case of therapy-refractory, progressive ependymoma with cerebrospinal as well as extraneural metastases, which allowed us for the first time to follow the stepwise accumulation of chromosome aberrations during disease progression. Genome-wide DNA copy-number analysis showed sequential deletions on chromosomes 1, 9, and 14 as well as a homozygous deletion of the CDKN2A locus, underscoring its role in tumor progression. Gradual loss at 1p36 was associated with loss of protein expression of the putative tumor suppressor gene AJAP1/SHREW1. In summary, this is the first report on acquired genomic aberrations in ependymoma over time pointing to novel candidate tumor suppressor genes. This analysis provides molecular insights into the chronology of genetic events in this case from initial localized tumor to widespread metastasized disease.
Genes Chromosomes and Cancer 12/2008; 48(3):229-38. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine whether a mixed population of immortalized human endometriosis epithelial and stromal cells is able to induce peritoneal endometriosis in nude mice.
Prospective experimental study. Human immortalized endometriosis epithelial and stromal cells were xenografted into ovariectomized nude mice. Macroscopically, the number of induced endometriosis-like lesions and their color were determined. Microscopically, histomorphology of endometriosis glands and their structure were analyzed, and comparisons were made with tissue from spontaneous endometriosis in women.
College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.
Seven ovariectomized nude mice.
Minimal invasive procedures were performed to administer estrogen pellets and transplant immortalized human endometriosis epithelial and stromal cells into nude mice.
Peritoneal endometriosis-like lesions induced in nude mice were characterized and compared with spontaneous peritoneal endometriosis in women.
Xenografts of human immortalized endometriosis epithelial and stromal cells into the peritoneal cavity of the recipient nude mice are able to proliferate, attach, invade, reorganize, and establish peritoneal endometriosis. Endometriosis glands at different stages of growth were present in induced endometriosis-like lesions. Proliferating cell nuclear antigen, metalloproteinase 2, estrogen receptor-alpha, cyclooxygenase-2, and prostaglandin E(2) receptors EP2 and EP4 proteins were expressed in both endometriosis glandular epithelial and stromal cells of the induced endometriosis-like lesions.
This xenograft model could be used as a potential experimental tool to understand the molecular and cellular aspects of the pathogenesis of endometriosis in humans.
Fertility and sterility 09/2008; 91(5 Suppl):2199-209. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The promoter of P receptor isoform B (PR-B) has recently been shown to be hypermethylated in endometriotic tissues, but one question remains as what causes the aberrant methylation. We show that sustained stimulation of an epithelial-like endometriotic cell line with tumor necrosis factor alpha stimulation for 30 days induced partial methylation in the promoter region of PR-B with concomitant reduction of PR-B expression.
Fertility and sterility 08/2008; 90(1):234-7. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signal peptides (SP) and transmembrane segments (TMS) ensure proper subcellular targeting and localization of proteins. Thus, understanding the molecular variability of this targeting information is essential. In this study, we functionally analyzed the predicted SP and the TMS of adherens junction protein, shrew-1 (Bharti et al. Novel membrane protein shrew-1 targets to cadherin-mediated junctions in polarized epithelial cells. Mol Biol Cell 2004:15:397). We used human secreted alkaline phosphatase (SEAP) as reporter protein. The SP of shrew-1 was able to functionally substitute for SEAP's intrinsic SP and was cleaved, indicating that it acts as a start-transfer signal and not a signal anchor. In turn, the TMS of shrew-1 functions as stop-transfer signal. Notably, clearly detectable plasma membrane localization is only achieved when the fusion protein contains both the SP and the TMS of shrew-1. In combination with the intrinsic SP from SEAP, the shrew-1 TMS is unable to promote stable plasma membrane localization. Hence, it may be assumed that this synergism between an SP and a TMS to mediate plasma membrane localization is essential for structural and/or functional integrity of shrew-1.