Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous... more Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous wound healing remains a significant medical challenge. A bioengineered skin that stimulates the body's natural regeneration capability is needed to address the current lack of treatment options. To this end, a biocompatible collagen wound matrix was developed using an electrochemical deposition fabrication process. The advanced collagen wound matrix has relatively high tensile strength compared to normal collagen matrix made by the heat gelation process and open porosity, and serves as an excellent platform for cellular growth and differentiation. Human adipose derived stem cells (hADSCs) were cultured on this collagen matrix and a co-culture system with primary keratinocytes and keratinocyte conditioned media was developed for differentiation of the hADSCs to keratinocyte-like cells. After fifteen days, hADSCs in co-culture began to exhibit a "cobblestone-like" morphology, indicating preliminary signs of differentiation to a keratinocyte-like cell. Based on morphological analysis at day 30, the co-culture with keratinocyte conditioned media system shows promising preliminary evidence of hADSC differentiation to a keratinocyte-like cell on an electrochemically aligned collagen wound matrix.
Functional profiling of CFTR-directed therapeutics offers the potential to provide significant be... more Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiX TM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (I CFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm 2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.
Scavenger receptor B1 (SR-B1) is a transmembrane protein, involved in tissue reverse cholesterol ... more Scavenger receptor B1 (SR-B1) is a transmembrane protein, involved in tissue reverse cholesterol transport and known as HDLs main receptor. Several studies have demonstrated that SR-B1 is also implicated in several other processes, such as bacteria and apoptotic cells recognition and regulation of intracellular antioxidant levels. Although this receptor is mainly localized in the liver and steroidogenic tissues, it has been shown that it is significantly expressed also in human skin, especially in the epidermis. Our purpose, by using 2D and 3D human keratinocytes cultures, was to investigate the role of SR-B1 in cutaneous tissue proliferation and differentiation under exogenous oxidative stress conditions. We have demonstrated that environmental stressors, such as ozone, air particles and cigarette smoke, downregulate SR-B1 expression affecting also keratinocytes ability to recover from wound scratch. In fact, SR-B1 knockdown reduced cell proliferation capacity and wound scratched knockdown keratinocytes presented lower nuclear Cyclin D1 protein levels. SR-B1 knockdown in tridimensional skin equivalents showed changes in stratum corneum thickness and this correlated with changes in its lipid distribution. Our findings suggest that SR-B1 might play an important role in keratinocytes proliferation and recovery from external insults, as well as in epidermal differentiation and lipid composition.
By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immuno... more By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.
Green fluorescent protein (GFP) has served as a versa-Remember-your existing TTO User Name and Pa... more Green fluorescent protein (GFP) has served as a versa-Remember-your existing TTO User Name and Password will not be valid, you must register with BioMedNet.
www.invitrogen.com FluoCells ® prepared slide #6 showing a fixed, permeabilized, and labeled munt... more www.invitrogen.com FluoCells ® prepared slide #6 showing a fixed, permeabilized, and labeled muntjac skin fibroblast. Mitochondria were labeled with anti-OxPhos Complex V inhibitor protein mouse IgG1 and visualized using orange-fluorescent Alexa Fluor ® 555 goat anti-mouse IgG. F-actin was labeled with green-fluorescent Alexa Fluor ® 488 phalloidin, and the nucleus was stained with TO-PRO®-3 iodide.
SNP Fingerprinting of parental and conditionally reprogrammed samples reveals homology across all... more SNP Fingerprinting of parental and conditionally reprogrammed samples reveals homology across all sample pairs. Sample phylogeny shows clear concordance with key SNPs, confirming sample identities. Phylogenic clustering was used to identify matched parental and conditionally reprogrammed (CR) samples. Figure S2. CR-PDX cells are amenable to in vitro chemosensitivity screening. CR-PDX cells were removed from CR conditions and treated with targeted agents at concentrations ranging from 0.01-10 μM. Staurosporine was used as a positive control and viability was assessed after 3 to 5 days (a) CR-OV0857F, (b) CR-LG0567F (c) CR-HLXF-036LN, (d) CR-HLXF-056. Figure S3. CR-PDX cells are amenable to gene knockdown studies using siRNA. CR-OV0857F cells were reverse transfected with indicated siRNAs for 120 h. Knockdown efficiency was confirmed by qPCR and the effect of knockdown on viability was assessed at the end of study. (PPTX 3628 kb)
Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclin... more Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclinical model to standard cell line xenografts. PDX models faithfully recapitulate the original patient genetic profile, gene expression patterns and tissue histology. Despite their benefits, PDX models are limited by their inherent variability, lower throughput and lack of growth in vitro. The ability to generate cell lines from PDX models would enable high throughput chemosensitivity screens, ex vivo genetic manipulation and the development of novel orthotopic models. Development of stable PDX cell lines remains a challenge due to murine stromal outgrowth, lineage commitment and limited differentiation potential. Conditional reprogramming (CR) cell technology is a novel cell culture system facilitating the generation of stable cultures without genetic manipulation (Liu, Am J Pathol, 2012). The success of CR cell technology is dependent upon the combination of feeder cell-derived factors and Rho Kinase (ROCK) inhibitor. CR cells, therefore, represent a new class of progenitor-like cells, distinct from the phenotype of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The purpose of this study was to identify the advantages, limitations and potential applications of CR technology for derivation of PDX explant cell lines. Early passage human lung and ovarian PDX tumors were cultured in CR conditions to create stable explant cell lines. Cell lines were established from 5/8 (63%) PDX tumors and were expanded over 6 months in culture with varying morphologies and growth kinetics. Due to normal outgrowth of murine stromal cells, early CR cultures contained mixed populations and required murine depletion to enrich for human cells. Key oncogenic mutations in a model of ovarian papillary serous adenocarcinoma were preserved in the enriched tumor cell population. While purified CR PDX cell lines were amenable to high throughput chemosensitivity screens, in vitro chemosensitivity did not consistently predict response in in vivo murine models. The CR PDX cell lines were additionally assessed for genetic manipulation and ability to form tumors in vivo. Collectively, these results demonstrate the applications of CR technology for the generation of stable explant cell lines from PDX models for preclinical studies. Citation Format: Alexandra Borodovsky, Travis J. McQuiston, Brian Dougherty, Ambar Ahmed, David Whitston, Daniel Stetson, Gretchen K. Hubbard, Sharon S. Challberg, Brian A. Pollok, Celina M. D’Cruz. Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 641.
Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located... more Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predi... more Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-impla...
The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of... more The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathwa...
Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous... more Despite advances in tissue engineering and regenerative medicine, skin regeneration and cutaneous wound healing remains a significant medical challenge. A bioengineered skin that stimulates the body's natural regeneration capability is needed to address the current lack of treatment options. To this end, a biocompatible collagen wound matrix was developed using an electrochemical deposition fabrication process. The advanced collagen wound matrix has relatively high tensile strength compared to normal collagen matrix made by the heat gelation process and open porosity, and serves as an excellent platform for cellular growth and differentiation. Human adipose derived stem cells (hADSCs) were cultured on this collagen matrix and a co-culture system with primary keratinocytes and keratinocyte conditioned media was developed for differentiation of the hADSCs to keratinocyte-like cells. After fifteen days, hADSCs in co-culture began to exhibit a "cobblestone-like" morphology, indicating preliminary signs of differentiation to a keratinocyte-like cell. Based on morphological analysis at day 30, the co-culture with keratinocyte conditioned media system shows promising preliminary evidence of hADSC differentiation to a keratinocyte-like cell on an electrochemically aligned collagen wound matrix.
Functional profiling of CFTR-directed therapeutics offers the potential to provide significant be... more Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiX TM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (I CFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm 2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.
Scavenger receptor B1 (SR-B1) is a transmembrane protein, involved in tissue reverse cholesterol ... more Scavenger receptor B1 (SR-B1) is a transmembrane protein, involved in tissue reverse cholesterol transport and known as HDLs main receptor. Several studies have demonstrated that SR-B1 is also implicated in several other processes, such as bacteria and apoptotic cells recognition and regulation of intracellular antioxidant levels. Although this receptor is mainly localized in the liver and steroidogenic tissues, it has been shown that it is significantly expressed also in human skin, especially in the epidermis. Our purpose, by using 2D and 3D human keratinocytes cultures, was to investigate the role of SR-B1 in cutaneous tissue proliferation and differentiation under exogenous oxidative stress conditions. We have demonstrated that environmental stressors, such as ozone, air particles and cigarette smoke, downregulate SR-B1 expression affecting also keratinocytes ability to recover from wound scratch. In fact, SR-B1 knockdown reduced cell proliferation capacity and wound scratched knockdown keratinocytes presented lower nuclear Cyclin D1 protein levels. SR-B1 knockdown in tridimensional skin equivalents showed changes in stratum corneum thickness and this correlated with changes in its lipid distribution. Our findings suggest that SR-B1 might play an important role in keratinocytes proliferation and recovery from external insults, as well as in epidermal differentiation and lipid composition.
By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immuno... more By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.
Green fluorescent protein (GFP) has served as a versa-Remember-your existing TTO User Name and Pa... more Green fluorescent protein (GFP) has served as a versa-Remember-your existing TTO User Name and Password will not be valid, you must register with BioMedNet.
www.invitrogen.com FluoCells ® prepared slide #6 showing a fixed, permeabilized, and labeled munt... more www.invitrogen.com FluoCells ® prepared slide #6 showing a fixed, permeabilized, and labeled muntjac skin fibroblast. Mitochondria were labeled with anti-OxPhos Complex V inhibitor protein mouse IgG1 and visualized using orange-fluorescent Alexa Fluor ® 555 goat anti-mouse IgG. F-actin was labeled with green-fluorescent Alexa Fluor ® 488 phalloidin, and the nucleus was stained with TO-PRO®-3 iodide.
SNP Fingerprinting of parental and conditionally reprogrammed samples reveals homology across all... more SNP Fingerprinting of parental and conditionally reprogrammed samples reveals homology across all sample pairs. Sample phylogeny shows clear concordance with key SNPs, confirming sample identities. Phylogenic clustering was used to identify matched parental and conditionally reprogrammed (CR) samples. Figure S2. CR-PDX cells are amenable to in vitro chemosensitivity screening. CR-PDX cells were removed from CR conditions and treated with targeted agents at concentrations ranging from 0.01-10 μM. Staurosporine was used as a positive control and viability was assessed after 3 to 5 days (a) CR-OV0857F, (b) CR-LG0567F (c) CR-HLXF-036LN, (d) CR-HLXF-056. Figure S3. CR-PDX cells are amenable to gene knockdown studies using siRNA. CR-OV0857F cells were reverse transfected with indicated siRNAs for 120 h. Knockdown efficiency was confirmed by qPCR and the effect of knockdown on viability was assessed at the end of study. (PPTX 3628 kb)
Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclin... more Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclinical model to standard cell line xenografts. PDX models faithfully recapitulate the original patient genetic profile, gene expression patterns and tissue histology. Despite their benefits, PDX models are limited by their inherent variability, lower throughput and lack of growth in vitro. The ability to generate cell lines from PDX models would enable high throughput chemosensitivity screens, ex vivo genetic manipulation and the development of novel orthotopic models. Development of stable PDX cell lines remains a challenge due to murine stromal outgrowth, lineage commitment and limited differentiation potential. Conditional reprogramming (CR) cell technology is a novel cell culture system facilitating the generation of stable cultures without genetic manipulation (Liu, Am J Pathol, 2012). The success of CR cell technology is dependent upon the combination of feeder cell-derived factors and Rho Kinase (ROCK) inhibitor. CR cells, therefore, represent a new class of progenitor-like cells, distinct from the phenotype of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The purpose of this study was to identify the advantages, limitations and potential applications of CR technology for derivation of PDX explant cell lines. Early passage human lung and ovarian PDX tumors were cultured in CR conditions to create stable explant cell lines. Cell lines were established from 5/8 (63%) PDX tumors and were expanded over 6 months in culture with varying morphologies and growth kinetics. Due to normal outgrowth of murine stromal cells, early CR cultures contained mixed populations and required murine depletion to enrich for human cells. Key oncogenic mutations in a model of ovarian papillary serous adenocarcinoma were preserved in the enriched tumor cell population. While purified CR PDX cell lines were amenable to high throughput chemosensitivity screens, in vitro chemosensitivity did not consistently predict response in in vivo murine models. The CR PDX cell lines were additionally assessed for genetic manipulation and ability to form tumors in vivo. Collectively, these results demonstrate the applications of CR technology for the generation of stable explant cell lines from PDX models for preclinical studies. Citation Format: Alexandra Borodovsky, Travis J. McQuiston, Brian Dougherty, Ambar Ahmed, David Whitston, Daniel Stetson, Gretchen K. Hubbard, Sharon S. Challberg, Brian A. Pollok, Celina M. D’Cruz. Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 641.
Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located... more Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predi... more Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-impla...
The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of... more The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathwa...
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