Stem cell markers in gastrointestinal cancer

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21095-21095
Author(s):  
M. Valladares-Ayerbes ◽  
S. Díaz Prado ◽  
V. Medina ◽  
P. Iglesias ◽  
B. Rodríguez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Gastrointestinal Cancer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Markers ◽  
Continuous Growth

21095 Background: Cancer cells with stem cells (CSC) properties have been identified in different tumors. It is suggested that CSC are responsible for the continuous growth of tumors, metastasis and drug-resistance. Markers for stem cells have been described. Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. ABCG2 transporter (MDR1) gene expression has been described as surrogate for the side-population phenotype. PTTG1 has also been recently identified as a component of the molecular signature of human (hu) ES cell-lines. Methods: Using Digital Northern we have demonstrated a significant tag counts for PTTG1 and reticulocalbin 2 (RCN2) in 11 huES cell-lines of the CGAP. The objective of our work has been to assess gene expression of these SC markers in a panel of new gastrointestinal cancer (GC) cells lines (CL) developed in our laboratory. Quantitative assessment was obtained by real-time PCR relative to normal bone marrow (BM), colonic mucosa and established cell-lines. GCCLs have been developed from ascitic fluid obtained of pancreatic carcinoma (MBQ-OJC1) and colon cancer (JJPF-OJC4, LCM-OJC5 and JAC-OJC6). GCCLs had been fully characterized by cytomorphology, epithelial and tumor markers (keratins, EGFR, EpCAM, p53), karyotype and tumor spheroids cultures. Results: Expression for ABCG2, Nanog, Oct4, PTTG1 and RCN2 were clearly detected in all the GCCL. Relative levels for each mRNA shown wide variety. For example, ABCG2 mRNA was highly expressed (2–26 fold) in colon cancer CL relative to BM. RCN2 was overexpressed (more than 2 x 102 fold) in 3 GCCL. Conclusions: Our results show that expressions of different “stemness” genes are maintained in cultured cancer cells. These data suggest that CSC are present in metastatic sites and can be maintained in continuous culture. We hypothesized that PTTG1 and RCN2 could be tested as a new cancer stem cells markers. No significant financial relationships to disclose.

226 CELL LINE-DEPENDENT GENE EXPRESSION PROFILES IN MOUSE EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
S. Mamo ◽  
J. Kobolak ◽  
S. Becker ◽  
M. Horsch ◽  
J. Beckers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Molecular Markers ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Es Cell

Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ � 129S1) blastocysts (Nagy et al. 1993 PNAS 90, 8424–8428) and HM-1 ESC established from an inbred strain (Selfridge et al. 1992 Somat. Cell Mol. Genet. 18, 325–336), as our earlier study showed performance variations between these cells. ES cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (GIBCO-Invitrogen, Carlsbad, CA, USA) supplemented with Na pyruvate (0.11% w/w; GIBCO-Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA), fetal bovine serum (15% v/v; HyClone, Logan, UT, USA), 1000 U mL-1 murine-LIF (Chemicon International, Temecula, CA, USA), and antibiotics (penicillin: 50 U mL-1, streptomycin: 50 �g mL-1; Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, D�sseldorf, Germany) procedures. Fifteen �g of total RNA each from the contrasting samples were used for reverse transcription and labeling with either Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer, added to the cDNA arrays (custom produced at GSF) containing over 21 000 sequences, and hybridized for 17 h at 42�C. The microarray results of 4 independent hybridizations were analyzed, and differentially regulated genes were identified. Finally, the results of 4 randomly selected genes were verified by real-time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (P < 0.01) between the 2 ES cell lines. Of these, 8 transcripts were up-regulated and the rest down-regulated in the HM-1 ES cells. Most of these genes were over-represented in important biological processes such as growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus, based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the 2 ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the 2 ES cell lines. This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No.070246), and Hungarian National Science Fund (OTKA T046171).

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

scholarly journals Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 771-784 ◽  
Author(s):  
Fariborz Izadyar ◽  
Francis Pau ◽  
Joel Marh ◽  
Natalia Slepko ◽  
Tracy Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Complex Mixture ◽  
Neural Cells ◽  
Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Selective PI3K inhibition by BKM120 and BEZ235 alone or in combination with chemotherapy in wild-type and mutated human gastrointestinal cancer cell lines.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 522-522 ◽  
Author(s):  
Markus Hermann Moehler ◽  
Annett Mueller ◽  
Erika Bachmann ◽  
Carl Christoph Schimanski ◽  
Peter Robert Galle
Keyword(s):  
Gastric Cancer ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Gastrointestinal Cancer ◽  
Human Colon ◽  
Signalling Pathways ◽  
Wild Type ◽  
Pi3k Inhibitors ◽  
Apoptotic Effects

522 Background: New targeted agents against tyrosine kinases expand the standard therapy in oncology. However, tumor resistance is still a challenge, particularly induced by mutations in growth-related signalling cascades. 20% and 10% of patients with human colon and gastric cancer carry PI3K mutations and do not react to receptor blocking therapies. Recently, selective tyrosine kinase inhibitors have been generated which block PI3K signalling pathways in tumor cells. Their therapeutically role has not yet been clarified. Methods: To define inhibitory and pro-apoptotic effects of the 2 PI3K inhibitors BEZ235 and BKM120 3 human colon cancer (HT-29, HCT-116, DLD-1) and 3 gastric cancer cell lines (NCI-n87, AGS, MKN-45) with different PIK3CA mutation status were used. First, viability, apoptosis and caspase assays were performed during incubation with inhibitors alone or combined with cytotoxic agents. Second, molecular consequences for cell cycle and the signalling pathways were analysed by defining the protein levels by FACS and Western blot. Results: Both PI3K inhibitors BEZ235 and BKM120 induced concentration dependently a significant reduction in viability and an increase in apoptotic death, while mutated cells reacted more sensitive to treatment. BKM120 had a higher efficiency than the dual PI3K/mTOR inhibitor BEZ235. BEZ235 alone caused a G1 arrest in tumor cells. In contrast, BKM120 induced a G2 shift in all gastrointestinal cancer cells. There was a clear downregulation in AKT signalling, and for BEZ235 an additional inhibition of mTOR pathway. Furthermore, BEZ235 caused synergistic induction of apoptosis combined with irinotecan in colon cancer. Combinations with 5-fluoruracil and the 2 substances induced additive apoptotic effects. Human gastric cancer cells were less sensitive to BEZ235 and BKM120. Conclusions: In general, we found higher pro-apoptotic effects for all cell lines and in special cases a better response of resistant mutant cells. Our data support the clinical development of these PI3K inhibitors BEZ235 and BKM120 as potential targeting agents for patients with different wild-type or mutated gastrointestinal cancer cells.

scholarly journals A Gene Expression–Based Screening System for Compounds Influencing Differentiation of Mouse Embryonic Stem Cells

2011 ◽  
Vol 17 (2) ◽  
pp. 140-151 ◽  
Author(s):  
Yuya Kunisada ◽  
Masanobu Shoji ◽  
Masaki Hosoya
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
High Throughput Screening ◽  
Adult Stem Cells ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Screening System ◽  
Pharmacologically Active

Low molecular weight compounds have been shown to be useful for controlling stem cells, and various high-throughput screening systems have been developed for identifying compounds that regulate the differentiation of stem cells. However, the effects of such compounds on stem cell differentiation are usually evaluated by assessing a single parameter, which is insufficient for proper monitoring of the cellular status. In this study, to classify a number of compounds, the authors established a gene expression–based screening system using mouse embryonic stem (ES) cells that monitored multiple parameters. ES cells were differentiated into three germ layers by embryoid body formation and then treated with the test compounds. Next, cellular changes were assessed by analyzing the expression of multiple genes with the multiplex quantitative reverse transcriptase polymerase chain reaction. By screening a library of pharmacologically active compounds with this system, the authors were able to classify 52 compounds that influenced the gene expression profile of ES cells. They also found that some compounds identified by screening could enhance osteogenic or adipogenic differentiation of human mesenchymal stem cells. These results indicate that the screening system is effective for identifying compounds involved in regulating the differentiation of both ES cells and adult stem cells.

scholarly journals Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758 ◽  
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

scholarly journals Investigation of MACC1 Gene Expression in Head and Neck Cancer and Cancer Stem Cells

2016 ◽  
Vol 39 (6) ◽  
pp. 77 ◽  
Author(s):  
Ebru Evran ◽  
Hilal Şahin ◽  
Kübra Akbaş ◽  
Sadik Çiğdem ◽  
Esra Gündüz
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Colon Cancer ◽  
Head And Neck Cancer ◽  
Cancer Stem Cells ◽  
Head And Neck ◽  
Cancer Cells ◽  
Neck Cancer ◽  
Positive Expression ◽  
Potential Biomarker

Purpose: By investigating the MACC1 gene (metastasis-associated in colon cancer 1) in cancer stem cells (CSC) resistant to chemotherapy and in cancer stem cells (CSC) resistant to chemotherapy and in cancer cells (CS) sensitive to chemotherapy we determineda steady expression in both types of cells in head and neck cancer. In conformity with the result we examined if this gene could be a competitor gene for chemotherapy. According to literature, the MACC1 gene shows a clear expression in head and neck cancer cells [1]. Here we examined MACC1 expression in CSC and investigated it as a possible biomarker. Methods: Our experiments were performed in the UT -SCC -74 in primary head and neck cancer cell line. We examined the MACC -1 gene expression by Real Time PCR from both isolated CSC and CS. Results: Expression of MACC -1 gene of cancer stem cells showed an two-fold increase compared with cancer cells. Based on the positive expression of MACC1 in both CS and CSC, this gene may serve as a potential biomarker in head and neck cancer. By comparing the results of this study with the novel features of MACC1, two important hypotheses could be examined. The first hypothesis is that MACC1 is a possible transcripton factor in colon cancer, which influences a high expression of CSC in head and neck and affects the expression of three biomarkers of the CSC control group biomarkers. The second hypothesisis is that the positive expression of MACC1 in patients with a malignant prognosis of tongue cancer, which belongs to head and neck cancer types, operates a faster development of CSC to cancer cells.

scholarly journals Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells

2018 ◽  
Vol 38 (11) ◽  
Author(s):  
Emilia Kuuluvainen ◽  
Eva Domènech-Moreno ◽  
Elina H. Niemelä ◽  
Tomi P. Mäkelä
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Colon Cancer ◽  
Cancer Cells ◽  
Therapeutic Targets ◽  
Cyclin Dependent Kinase ◽  
Colon Cancer Cells ◽  
Concomitant Decrease ◽  
Oncogene Activation ◽  
Myc Gene

ABSTRACT In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by β-catenin, which has previously been shown to associate with MED12. Importantly, β-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.

Expression of Functional Toll-Like Receptor 2 on Mouse Embryonic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4746-4746
Author(s):  
Tammi Taylor ◽  
Wilbert Derbigny ◽  
Young-June Kim ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Es Cells ◽  
Mrna Level ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Toll Like Receptor ◽  
Undifferentiated State ◽  
Mes Cells

Abstract Embryonic stem (ES) cells have the capacity to produce all cell types of the body. Understanding murine ES (mES) cell proliferation, survival, differentiation, and self renewal will enhance knowledge of developmental biology and essential use of ES cells. Recently, Toll Like Receptor (TLR) activation has been shown by others to play a role in influencing the differentiation of hematopoietic stem cells. Previous studies have also shown that TLR activation prevents mesenchymal stem cell differentiation into adipocytes, chondrocytes, and osteocytes and plays a role in bone repair. We hypothesized that certain TLR’s would be expressed on mES cells and that the ligands for these expressed TLR’s would induce functional activity in the mESC’s. Therefore, we wanted to determine if TLRs are expressed on mES cells and if so, are they functional. Three different mES cell lines (R1, CGR8, and E14) were used to determine if TLRs are expressed at the mRNA level using primers for murine TLR1-9 mRNA. We found that TLR’s 1, 2, 3, 6, and 9 were expressed at the mRNA level, but TLR’s 4, 5, 7, and 8 were not. Based on the availability of antibodies to TLR’s, and using flow cytometry, we found expression of TLR2 but not TLR 4 on the surface of all three mES cell lines. TLR ligands were used to treat mES cells in the presence of leukemia inhibitory factor (LIF) for an hour. Activation of TLR2 by its ligand Pam3Cys, a synthetic tri-acyl lipoprotein, on mES cells induced NF-κβ nuclear translocation when compared to ES cells not stimulated with TLR ligands. LPS, the ligand for TLR4 did not induce NF-κβ nuclear translocation on ES cells, consistent with lack of cell surface expression of TLR4 on mES cells. TLR expression and TLR ligand interaction were not associated with changes in the morphology of the mES cells or expression of Oct-4, SSEA-1, KLF-4, or Sox-2, markers for maintenance of the undifferentiated state of mES cells. This suggests that the cells remain in an undifferentiated state even after TLR activation by Pam3Cys in the presence of LIF. Thus our study has identified functionally active TLR2 on the surface of mES cells, information that may be of use to further defining a role for TLR’s on ES cells, and for manipulation of other ES cell functions.

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