Biomedical subjects

S J Gendler

Publications and source records attributed to S J Gendler.

At least 37 records · Page 2Linked to original sources

PubMed1999

Evaluation of 5-aminosalicylic acid (5-ASA) for cancer chemoprevention: lack of efficacy against nascent adenomatous polyps in the Apc(Min) mouse.

Recent experimental and epidemiological evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in the prevention of colorectal cancer. However, the toxicity associated with the long-term use of most classical NSAIDs has limited their usefulness for the purpose of cancer chemoprevention. Inflammatory bowel disease (IBD) patients, in particular, are sensitive to the adverse side effects of NSAIDs, and these patients also have an increased risk for the development of intestinal cancer. 5-Aminosalicylic acid (5-ASA) is an anti-inflammatory drug commonly used in the treatment of IBD and may provide protection against the development of colorectal cancer in these patients. To directly evaluate the ability of 5-ASA to suppress intestinal tumors, we studied several formulations of 5-ASA (free acid, sulfasalazine, and Pentasa) at multiple oral dosage levels [500, 2400, 4800, and 9600 parts/million (ppm)] in the adenomatous polyposis coli (Apc) mouse model of multiple intestinal neoplasia (Min). Although the ApcMin mouse is not a model of colitis-associated neoplasia, it is, nonetheless, a useful model for assessing the ability of anti-inflammatory agents to prevent tumor formation in a genetically preinitiated population of cells. We used a study design in which drug was provided ad libitum through the diet beginning at the time of weaning (28 days of age) until 100 days of age. We included 200 ppm of piroxicam and 160 ppm of sulindac as positive controls, and the negative control was AIN-93G diet alone. Treatment with either piroxicam or sulindac produced statistically significant reductions in intestinal tumor multiplicity (95% and 83% reductions in tumor number, respectively; P < 0.001 versus controls). By contrast, none of the 5-ASA drug formulations or dosage levels produced consistent dose-progressive changes in polyp number, distribution, or size, despite high luminal and serum concentrations of 5-ASA and its primary metabolite N-acetyl-5-ASA. Thus, 5-ASA does not seem to possess direct chemosuppressive activity against the development of nascent intestinal adenomas in the ApcMin mouse. However, because intestinal tumor development in the ApcMin mouse is driven by a germline mutation in the Apc gene rather than by chronic inflammation, we caution that these findings do not definitively exclude the possibility that 5-ASA may exert a chemopreventive effect in human IBD patients.

Adenomatous Polyps↗

PubMed1999

Bacterial conjunctivitis in Muc1 null mice.

PURPOSE: In contrast to wild-type mice, genetically engineered Mucin1 (Muc1) null animals display a marked propensity for development of blepharitis and conjunctivitis. Molecular approaches confirmed the presence of Muc1 mRNA and protein in the conjunctival tissue of wild-type mice and identified the bacterial species in Muc1 null symptomatic mice. METHODS: Muc1 null animals housed in a conventional facility were examined for visually apparent inflammation of the eye and surrounding tissue. Blood taken from overtly affected animals was assayed for antibodies to common murine viral agents. Swabs of infected eyes and whole eye preparations were used to detect and speciate bacterial pathogens. Frozen sections of whole eye, lid margin, and Harderian gland were immunostained with antibodies to Muc1 and cytokeratin 14, both epithelial cell markers. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed on RNA isolated from conjunctiva and Harderian gland of wild-type mice to compare relative levels of transcript. RESULTS: Student's unpaired t-test performed on the eye inflammation frequency of Muc1 null mice confirmed a statistical significance (P < 0.01) when compared to wild-type background animals housed in the same room. Analysis of blood samples from affected Muc1 null animals detected no common murine viral pathogens. Bacterial analysis of conjunctival swabs and whole eye preparations demonstrated the presence of coagulase-negative Staphylococcus, Streptococcus type alpha, and Corynebacterium group G2. Muc1 antibody staining of wild-type sections revealed the presence of Muc1 on conjunctival goblet and non-goblet cells and on the epithelium of the Harderian gland. Serial sections stained with cytokeratin 14 antibody confirmed the epithelial nature of cells expressing the Muc1 protein. RNA from conjunctiva and Harderian gland subjected to RT-PCR and northern blot analysis showed an abundance of Muc1 transcript in these tissues. CONCLUSIONS: Muc1 mRNA and protein are present in murine conjunctival and Harderian gland epithelia. Animals lacking Muc1 mRNA and protein are predisposed to developing eye inflammation when compared to wild-type animals with an intact Muc1 gene. Muc1 appears to play a critical protective role at the ocular surface, presumably by acting as a barrier to infection by certain bacterial strains.

Animals↗

PubMed1998

Cystic fibrosis mice lacking Muc1 have reduced amounts of intestinal mucus.

Normally a thin layer of mucus covers the surface of the gastrointestinal tract protecting the epithelial cells from their environment. In cystic fibrosis (CF), mucus accumulation is abnormally high, resulting in severe intestinal obstruction. The major structural components of mucus are large mucin glycoproteins. We determined specific mucin RNA and protein expression in the gastrointestinal tract of inbred CF transmembrane conductance regulator (CFTR) knockout (CF) mice and correlated expression with histological analyses of tissues. Mucins were detected histochemically using general carbohydrate stains and specific mucin antibodies. Mucin RNA levels were determined by reverse transcription-PCR. Comparisons were made between CF mice and control siblings, all maintained on a liquid diet after weaning. Analyses of the mucins Muc2, Muc3, and Muc5ac showed lower levels of RNA expression in the CF mice and similar levels of protein. Significantly, there was a sixfold increase in Muc1 RNA expression in the colon of the CF mouse and a moderate increase in Muc1 protein. Further, CF mice lacking Muc1 exhibited greatly diminished intestinal mucus obstruction when compared with Muc1- expressing CF mice and had better survival on solid food. We suggest that Muc1 plays an important role in the mucus obstructions observed in the gastrointestinal tract of the CFTR knockout mouse.

Animals↗

PubMed1998

CD4+ lymphocytes provide MUC1-specific tumor immunity in vivo that is undetectable in vitro and is absent in MUC1 transgenic mice.

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC .Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.

Adoptive Transfer↗

PubMed1998

Estrogen receptor does not directly regulate the murine Muc-1 promoter.

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.

Animals↗

PubMed1998

Genetic modulation of neu proto-oncogene-induced mammary tumorigenesis.

Modulation of oncogene-induced carcinogenesis by secondary mutation or genetic background may be an important factor in determining the expression of the tumor phenotype. We have investigated the role of loss of function mutations and strain-specific genetic elements in the modulation of oncogene-induced breast cancer using a murine model. FVB female mice transgenic for the rat neu proto-oncogene [mouse mammary tumor virus (MMTV)-neu] developed mammary tumors between 7 and 12 months of age, whereas FVB x C57Bl/6 (F1) MMTV-neu mice had tumor latencies greater than 18 months. The expression level of the neu transgene was equivalent in tumor tissue from both FVB and F1 mice. Furthermore, increased tumor latency did not appear to be associated with a decrease in expression of the neu transgene in the normal mammary gland of F1 mice because immunohistochemical staining for neu expression in the mammary glands of 3-month-old virgin female mice revealed similar levels of protein expression in FVB and F1 animals. When F1 animals were backcrossed one generation onto the FVB strain ([FVB x B6] F1 x FVB), a subset of the resulting offspring developed tumors with a latency equivalent to that of the pure-strain FVB mice. Statistical analysis of the genetic variability in mammary tumor latency indicated that approximately three independent genes were involved in the latency effect. Interestingly, when tumor growth rates were compared in these same animals, F1 mice had significantly faster tumor growth rates compared with FVB mice.

Animals↗

PubMed1998

Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells.

Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumor-associated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinoma-associated antigen in MUC1 transgenic mice unresponsive to MUC1 antigen. Immunization of adult MUC1 transgenic mice with irradiated MUC1-positive tumor cells was unsuccessful in reversing unresponsiveness to MUC1. By contrast, fusions of dendritic cells with MUC1-positive tumor cells induced cellular and humoral immunity against MUC1. Immunization with the dendritic cell fusions that express MUC1 resulted in the rejection of established metastases and no apparent autoimmunity against normal tissues. These findings demonstrate that unresponsiveness to the MUC1 tumor-associated antigen is reversible by immunization with heterokaryons of dendritic cells and MUC1-positive carcinoma cells.

Adult↗

PubMed1998

Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model.

The human epithelial mucin, MUC1, is a large transmembrane glycoprotein that is expressed on most simple epithelia. It is overexpressed and aberrantly glycosylated on many human epithelial tumors, including more than 90% of human breast cancers. MUC1 is of interest as an immunotherapy target because patients with breast, ovarian, and pancreatic cancers have T lymphocytes in their tumor-draining lymph nodes that can be induced to recognize and lyse MUC1-expressing tumor cells. We have produced a transgenic mouse model that expresses the human MUC1 molecule on an inbred C57Bl/6 background to investigate the effect of endogenous expression of MUC1 on the ability of mice to generate antitumor immunity to MUC1-expressing tumors. Transgenic mice expressed the human transgene in a pattern and level consistent with that observed in humans. Transgenic mice were tolerant to stimulation by MUC1 as evidenced by the ability of MUC1-expressing tumor cells to grow in these mice, whereas MUC1-expressing cells were eliminated from wild-type mice. Moreover, transgenic mice immunized with MUC1 peptides failed to exhibit immunoglobulin class switching to the IgG subtypes. These data suggest that endogenous expression of MUC1 protein by MUC1 transgenic mice induces T-cell tolerance to stimulation by MUC1. The transgenic mice will provide a useful model to investigate the mechanisms that regulate immunological tolerance to tumor antigens and will facilitate the investigation of anti-MUC1 immunotherapy formulations.

Animals↗

PubMed1998

A microsatellite within the MUC1 locus at 1q21 is altered in the neoplastic cells of breast cancer patients.

Paired DNA samples from the neoplastic and nonneoplastic cells of 118 patients with the sporadic, nonfamilial form of breast cancer were analyzed for evidence of genetic alteration at a polymorphic microsatellite mapped to intron 6 within the MUC1 gene at 1q21. Two other microsatellite loci, D1S104 and APO-A2, which also map to 1q21, were analyzed as well. The frequency of alteration at the microsatellite within the MUC1 locus was significantly higher than D1S104 or APO-A2 (P < 0.001). Analysis by Southern blotting of the VNTR region of the MUC1 gene revealed an amplification of one allele in 34 of 54 informative cases (63%). There was no significant association between these alterations and alterations of the microsatellite within the same locus, suggesting independent mechanisms were responsible for the genetic changes. Microsatellite loci D17S579 at 17q21, the site of the BRCA1 gene, and D18S34 at 18q21-qter, the deleted in colorectal cancer locus, were also analyzed by PCR. Alterations at D17S579 and D18S34 were detected in 18.8% and 6.2% of patients, respectively (P < 0.001, and P < 0.1 relative to the frequency of alteration at D1S104 or APO-A2). A previously described polymorphism of hMSH2 was altered in 16.4% of cases.

Breast Neoplasms↗

PubMed1998

Abnormal heart rate regulation in GIRK4 knockout mice.

Acetylcholine (ACh) released from the stimulated vagus nerve decreases heart rate via modulation of several types of ion channels expressed in cardiac pacemaker cells. Although the muscarinic-gated potassium channel I(KACh) has been implicated in vagally mediated heart rate regulation, questions concerning the extent of its contribution have remained unanswered. To assess the role of I(KACh) in heart rate regulation in vivo, we generated a mouse line deficient in I(KACh) by targeted disruption of the gene coding for GIRK4, one of the channel subunits. We analyzed heart rate and heart rate variability at rest and after pharmacological manipulation in unrestrained conscious mice using electrocardiogram (ECG) telemetry. We found that I(KACh) mediated approximately half of the negative chronotropic effects of vagal stimulation and adenosine on heart rate. In addition, this study indicates that I(KACh) is necessary for the fast fluctuations in heart rate responsible for beat-to-beat control of heart activity, both at rest and after vagal stimulation. Interestingly, noncholinergic systems also appear to modulate heart activity through I(KACh). Thus, I(KACh) is critical for effective heart rate regulation in mice.

Adenosine↗

PubMed1998

The biological role of mucins in cellular interactions and immune regulation: prospects for cancer immunotherapy.

Among the human mucins, MUC1 is unique in its cell-surface transmembrane expression and its apparent signal-transduction functions. The high expression of MUC1 on many human cancers makes it an attractive target for immunotherapy. Immunization of human cancer patients with MUC1 peptides has resulted in the generation of both anti-MUC1 antibody and cytotoxic T lymphocyte responses. Recently, a novel immunoregulatory role for MUC1 has been suggested by experiments demonstrating that soluble MUC1 induces T-cell unresponsiveness, and that T cells appear to express and secrete MUC1 following their activation. MUC1 is an apparent paradox, having both adhesive and antiadhesive functions, and immunostimulatory and immunosuppressive activities.

Amino Acid Sequence↗

PubMed1998

Mimic 1 of MUC1.

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Animals↗

PubMed1997

Histological studies of gene-ablated mice support important functional roles for natural killer cells in the uterus during pregnancy.

Maternal lymphocytes having a large and granulated morphology accumulate at healthy implantation sites in normal mice. Insight into the functions of these cells has come from a previous study of two independent lines of mice deficient in natural killer (NK) cells. In pregnant Tg epsilon 26 mice, vascular pathology was found that led to the major complications of either fetal death or intrauterine growth retardation. In pregnant p56lck null x IL-2R beta null mice, extensive distension of the decidua was observed that separated the placenta from the myometrium and appeared to be interstitial edema. To strengthen assignment of uterine large granulated lymphocytes to the NK cell lineage and to understand which aspects of NK cell biology may be important for a uterine-based, pregnancy-associated subset, mid-gestation implantation sites from a new series of mice having gene deletions which alter NK cells (IL-2R gamma null, Stat4 null, IL-12 p40 null, beta 7 integrin null and Muc-1 null) have been examined histologically. The findings support the assignment of pregnancy-associated large granulated cells of mice to the NK cell lineage and suggest that the primary functions of these tissue-based NK cells are to support normal development of the decidua and/or its vasculature using pathways that involve IL-12 mediated signal transduction.

Animals↗

PubMed1997

Loss of heterozygosity analysis in primary mammary tumors and lung metastases of MMTV-MTAg and MMTV-neu transgenic mice.

Loss of heterozygosity (LOH) analysis has been used in many types of human cancer to localize putative tumor suppressor genes important in carcinogenesis. However, this approach has only recently been applied to transgenic mouse tumor models, which offer greater opportunity for detailed molecular genetic analysis of tumor initiation and progression. To explore the possible role of secondary genetic events in transgenic mouse mammary tumor development, we performed microsatellite-based allelotypes on primary mammary adenocarcinomas and lung metastases arising in mice transgenic for the polyomavirus middle T antigen under the control of the mouse mammary tumor virus promoter/enhancer (MMTV-MTAg mice) and on primary mammary adenocarcinomas arising in mice transgenic for the neu proto-oncogene (MMTV-neu mice). We examined a total of 80 microsatellite loci distributed throughout the mouse genome for LOH and observed high rates of specific chromosomal loss but very low rates of background allelic loss in these tumors. For the MMTV-MTAg mice, no individual chromosomes showed rates of LOH significantly above the background rates. For MMTV-neu mice, markers on chromosome 4 showed LOH in 82% of mammary tumors, whereas markers on chromosome 3 showed loss in 29% of tumors. These data suggest that the middle T antigen transgenic mice do not undergo whole chromosome loss or large genomic deletions as common mechanisms of tumor formation and that chromosomes 3 and 4 may contain tumor suppressor gene loci that play important roles in the development of neu-mediated mouse mammary tumors.

Adenocarcinoma↗

PubMed1997

Partial structure, chromosome localization, and expression of the mouse Icln gene.

The molecular identities of most volume-regulated ion channel proteins and putative regulatory elements are currently unknown. Recently, a role for a nucleotide-sensitive chloride conductance regulator, ICln, in the function of a ubiquitous volume-regulated chloride channel has been suggested. Here, we report the cloning of a fragment of the mouse Icln gene and identification of probable Icln pseudogenes. The functional Icln gene was mapped independently to human chromosome 11q13.5-q14 and mouse chromosome 7 (50.3 cM). ICln mRNA was shown to be abundantly expressed and evenly distributed in all mouse tissues examined and at four stages of embryonic development, consistent with the proposed role of ICln in the regulation of a ubiquitous chloride channel.

Amino Acid Sequence↗

PubMed1997

Partial structure, chromosome localization, and expression of the mouse Girk4 gene.

The G protein-gated potassium channel IKACh constitutes part of a signaling pathway that mediates the negative chronotropic and inotropic effects of acetylcholine on cardiac physiology. Similar or identical ion channels regulate the excitability of many neurons in response to neurotransmitters. IKACh is composed of two homologous subunits, GIRK1 and GIRK4. Here we describe a partial genomic structure of the mouse Girk4 gene. Two exons containing the complete protein-coding sequence were identified. Girk4 was mapped to mouse chromosome 9 (13 cM), consistent with the mapping of human GIRK4 to chromosome 11q23-ter. GIRK4 mRNA was found mainly in mouse heart, with trace levels detected in brain, kidney, lung, and spleen. No detectable levels were observed in skeletal muscle, liver, and testis. The onset of GIRK4 mRNA expression in the developing mouse occurs between Embryonic Days 7 and 11, consistent with the appearance and function of the mouse heart.

Animals↗

PubMed1996

Synthesis and intracellular trafficking of Muc-1 and mucins by polarized mouse uterine epithelial cells.

Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 +/- 1 and 16.5 +/- 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 +/- 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 +/- 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.

Animals↗

PubMed1996

The epithelial mucin MUC1 contains at least two discrete signals specifying membrane localization in cells.

The MUC1 gene product (PEM, polymorphic epithelial mucin) is a cell-associated glycoprotein expressed on the apical surface of most simple secretory epithelia. The transmembrane and cytoplasmic domains of MUC1 have been shown to be highly conserved between mammalian species, and it has been shown that this molecule interacts with the actin cytoskeleton. Apical targeting signals in polarized cells have yet to be defined. The mechanism by which MUC1 is targeted and maintained on the apical surface is not known; correct localization, however, would be predicted to be crucial for function. In order to determine which domains of MUC1 were important for this localization, mutational analysis of the protein was undertaken. Using cytoplasmic tail deletion mutants, fusion proteins of MUC1 and CD2, and site-directed mutagenesis, it could be shown that MUC1 appeared to contain at least two motifs involved in apical localization. The first was located in the extracellular domain and was sufficient to confer apical localization on the fusion protein. The second was the Cys-GlnCys (CQC) motif at the junction of the cytoplasmic and transmembrane domains. This sequence was necessary for surface expression. These results suggest that MUC1 contains two discrete motifs important in its apical localization.

Amino Acid Sequence↗