Reinhard Kelsch

Increased expression of the adult stem cell marker Musashi‐1 in endometriosis and endometrial carcinoma

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA‐binding protein Musashi‐1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi‐1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi‐1 mRNA expression in the endometrium compared to the myometrium. Musashi‐1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi‐1 with its molecular target Notch‐1 and telomerase. In proliferative endometrium, the proportion of Musashi‐1‐positive cells in the basalis layer was significantly increased 1.5‐fold in the stroma, and three‐fold in endometrial glands compared to the functionalis. The number of Musashi‐1 expressing cell groups was significantly increased (four‐fold) in proliferative compared to secretory endometrium. Musashi‐1 expressing stromal cell and cell group numbers were significantly increased (five‐fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi‐1‐expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma. Copyright

Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

Flow cytometry in cancer stem cell analysis and separation

In recent years, a special type of cancer cell—the cancer stem cell (CSC)—has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell‐specific activity of certain ATP‐binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell‐specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry‐based sorting principles and future flow cytometric applications for CSC analysis are also discussed.

Targeting of syndecan-1 by microRNA miR-10b promotes breast cancer cell motility and invasiveness via a Rho-GTPase- and E-cadherin-dependent mechanism.

microRNAs are small endogenous noncoding RNAs, which post‐transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan‐1, a predicted target of the oncomiR miR‐10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR‐10b and syndecan‐1, MDA‐MB‐231 and MCF‐7 breast cancer cells were transiently transfected with pre‐miR‐10b, syndecan‐1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time‐lapse video microscopy. miR‐10b overexpression induced post‐transcriptional downregulation of syndecan‐1, as demonstrated by quantitative real‐time PCR (qPCR), flow cytometry, and 3′UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan‐1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan‐1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan‐1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan‐1‐deficient cells revealed upregulation of ATF‐2, COX‐2, cadherin‐11, vinculin, actin γ 2, MYL9, transgelin‐1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E‐cadherin, CLDN1, p21WAF/CIP, cyclin‐dependent kinase 6, TLR‐4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin‐2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase‐dependent motility of syndecan‐1‐depleted cells, which displayed increased filopodia formation. We conclude that syndecan‐1 is a novel target of the oncomiR miR‐10b. Rho‐GTPase‐dependent modulation of cytoskeletal function and downregulation of E‐cadherin expression are identified as relevant effectors of the miR‐10b‐syndecan‐1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

Characterization of endometrial mesenchymal stem-like cells obtained by endometrial biopsy during routine diagnostics

Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of CD146, CD105, CD90, CD73, MSI1, NOTCH1, and SOX2; and absence of CD34 and CD14 expression. We conclude that adult stem cells are present in endometrial biopsies performed in a routine clinical setting, offering new large-scale approaches for future research, diagnostics, and therapies involving adult stem cells.

The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1

The RNA‐binding protein Musashi‐1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch‐1 signaling pathway. Musashi‐1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi‐1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase‐1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi‐1 expression is 20.8‐fold upregulated in SP+ compared to SP‐ and equally distributed between ALDH+ and ALDH‐ cell pools. siRNA‐mediated knockdown of Musashi‐1 mRNA expression lead to an altered expression of the signaling receptor Notch‐1 and its downstream targets, the transcription factor Hes‐1 and the cell cycle regulators p21WAF1/CIP1 and cyclin B1, as determined by Western blotting and quantitative real‐time PCR. Flow cytometric and ELISA analyses revealed that Musashi‐1‐mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell‐like properties. Musashi‐1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness‐related factors Notch‐1, Hes‐1 and p21WAF1/CIP1, thus emerging as a novel future target for endometrial carcinoma therapy.

Syndecan-1 (CD138) modulates triple-negative breast cancer stem cell properties via regulation of LRP-6 and IL-6-mediated STAT3 signaling.

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.

Accelerated kidney transplant rejection and hypertensive encephalopathy in a pediatric patient associated with antibodies against angiotensin type 1 receptor and HLA class II.

This work was supported by the University of Nebraska Medical Center Research Support Fund. J.T.L. has received money for support from Merck & Co., Inc. to study sitagliptin in a subsequent study. All other authors declare no conflicts of interest. Address correspondence to: James T. Lane, M.D., Division of Endocrinology and Diabetes, Department of Internal Medicine, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, 1000 N. Lincoln Blvd., Suite 2900, Oklahoma City, OK 73104-3252. E-mail: james-lane@ouhsc.edu J.T.L. participated in the design, and implementation of the study, participated in the recruitment and clinical care of patients, completed data acquisition, statistical analysis, and manuscript preparation. D.E.O. participated in statistical analysis, data acquisition, and review of the manuscript. C.E.H participated in the recruitment of patients, clinical follow-up within the clinical research center, data acquisition, and review of the manuscript. D.S.C. participated in the design of the study and review of the manuscript. L.E.W. performed renal transplants and administered clinical care. R.B.S. performed renal transplants, administered clinical care, participated in the design and implementation of the study, recruited patients, and reviewed the manuscript. Received 21 July 2011. Accepted 24 August 2011. Copyright

Establishing Reference Intervals for Sex Hormones on the Analytical Platforms Advia Centaur and Immulite 2000XP

Reliable reference intervals for sex hormones are indispensable in evaluations of the hypothalamo-pituitary-gonadal axis. This study established reference intervals for estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, and prolactin with the immunoassay platforms Advia Centaur and Immulite 2000XP (Siemens Healthcare, Germany). We recruited healthy men (n=220), women in the follicular (n=139) or luteal (n=87) phases of the menstrual cycle, and postmenopausal women (n=103). Data was analyzed according to CLSI EP28-A3c guidelines. Although reference intervals established with both platforms showed good agreement with ranges quoted by the assay manufacturer, two discrepancies were noted. First, intervals for prolactin in women were influenced by hormonal status, and the partition analysis supported their separation into subgroups based on menstrual cycle. Second, the upper limit for estradiol in the follicular phase was nearly a half of that provided by the manufacturer. This discrepancy was attributed to the stringent definition of the follicular phase (consistently set at days 3-5 after menstruation onset). Our findings suggest that reference values for prolactin should both be gender specific and account for menstrual cycle phase. The results also emphasize that clear-cut selection criteria are required when assembling populations for establishing endocrine reference intervals.

Telomere Length Is Not Related to Established Cardiovascular Risk Factors but Does Correlate with Red and White Blood Cell Counts in a German Blood Donor Population

Telomere length (TL) is considered a marker of biological aging and has been associated with the presence of various coronary risk factors in patients. Much less is known about the relationships between TL and classic coronary risk factors in other populations. We measured TL in peripheral blood leukocytes of 343 middle-aged blood donors (mean age 40.2 ± 12.4 years; 201 men, 142 women) using quantitative polymerase chain reaction. Median TL was 0.86 (range: 0.48–1.85) relative TL units. In linear regression analyses with natural log-transformed T to S ratio as the dependent variable, there was a significant association with age (per year: beta = -0.007, p<0.001) and sex (males vs. females: beta = 0.075, p = 0.007) with longer telomeres in men. After adjusting for these two variables, we observed no association of TL with classic coronary risk factors including cholesterol (p = 0.36), triglyceride (p = 0.09), HDL-cholesterol (p = 0.26), LDL-cholesterol (p = 0.36), smoking (p = 0.97), and personal (p = 0.46) or family history (p = 0.63) of cardiovascular disease. However, we did find a significant positive association with white (p = 0.011) and red blood cell count (p = 0.031), hemoglobin (p = 0.014) and hematocrit (p = 0.013); we also found a borderline positive association with thrombocytes (p = 0.074). Positive associations remained significant for hemoglobin (p = 0.017), hematocrit (p = 0.023), and leukocytes (p = 0.009) in a subgroup with no reported vascular disease; associations were of borderline significance for erythrocytes (p = 0.053) and thrombocytes (p = 0.088) in this subgroup. The data do not support the concept that classic coronary risk factors contribute to telomere attrition in a blood donor population. However, telomere attrition may be a marker for reduced proliferation reserve in hematopoietic progenitor cells.