OBJECTIVEThis study aims to investigate the expression and relationship of CD44 and CD133 in normal oral mucosa, oral potentially malignant disorder (OPMD), and oral squamous cell carcinoma (OSCC). This work also analyzes the relationship between such expression and clinical factors. This study intends to evaluate the clinical value of using CD44 and CD133 as indices to evaluate the carcinogenic potential of OPMD.

METHODSClinical data from 60 patients with OPMD, 60 patients with OSCC, and 10 cases of normal oral mucosa were analyzed. Double immunohistochemical analysis was applied to investigate the expression of CD44 and CD133 in paraffin sections of normal oral mucosa, OPMD, and OSCC tissues. Subsequently, the relationships between such expression and clinical factors were analyzed.

RESULTSThe positive rates of CD44 expression in the normal oral mucosa, OPMD, and OSCC tissues were 100.00%, 96.67%, and 71.67% (P<0.05), respectively. Meanwhile, the positive rates of CD133 expression in the normal oral mucosa, OPMD, and OSCC tissues were 0.00%, 35.00%, and 63.33% (P<0.05), respectively. The expression of CD44 and CD133 was found to be correlated (P<0.05). Such expression was related to the clinical stages and lymphatic metastasis of OSCC (P<0.05).

CONCLUSIONSCD44 and CD133 can be used individually as clinical indices to evaluate the carcinogenic potential of OPMD.
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AC133 Antigen ; Carcinogenesis ; Carcinoma, Squamous Cell ; Humans ; Hyaluronan Receptors ; Lymphatic Metastasis ; Mouth Mucosa ; Mouth Neoplasms

OBJECTIVETo study the expansion potential of megakaryocyte progenitor cells (MPC) from human placenta tissue CD133+ (PT-CD133+) cells.

METHODSPT-CD133+ cells were purified from mononuclear cells (MNC) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPC. At day 7, 10 and 14, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigens expression during ex-vivo expansion were analyzed by flow cytometry (FCM). PT-CD133+ cells at different expansion time were collected and cultured in collagen semisolid medium for colony forming units-megakaryocyte (CFU-MK) assay.

RESULTSPT-CD133+ cells could be optimally expanded at day 7 by 13 +/- 2 fold increase in serum-free liquid culture systems and the total cell number was expanded by 160 fold at day 14. With the expansion time going on, the expression of CD133, CD34 decreased and that of CD41 increased. The expanded megakaryocytes were immature and no sign of platelet formation. Both PT-CD133+ cells before and after expansion could form CFU-MK, the total number of CFU-MK peaked at day 10 of expansion by 54 +/- 10 fold increase.

CONCLUSIONHuman PT-CD133+ cells have a high capacity of MPC expansion, 10 days culture could give rise to the maximum number of CFU-MK.

AC133 Antigen ; Antigens, CD ; Cell Differentiation ; Cells, Cultured ; Female ; Glycoproteins ; Humans ; Megakaryocyte Progenitor Cells ; cytology ; Peptides ; Placenta ; cytology ; Pregnancy

OBJECTIVETo review the leading roles of glioma stem cells (GSCs) and their sophisticated interactions with other cells in the tissue remodeling process of gliomagenesis.

DATA SOURCESPublished articles about assessing GSCs in tumor initiation, progression, and multiple interactions with other cells in the special microenvironment were selected using PubMed. The search terms were "glioma stem cells", "tumorigenesis", and "microenvironment".

STUDY SELECTIONArticles regarding the tissue remodeling of GSCs in gliomagenesis were selected.

RESULTSGSCs exhibit enhanced tumor-initiating ability, could reestablish tumor, and were resistant to radiotherapy and chemotherapy. Studying the role of GSCs in gliomagenesis helps to develop targeting therapy against GSCs, which seems to be a cure for gliomas. However, sophisticated interactions between GSCs and their local microenvironment during tumor remodeling, including integrating with partially differentiated tumor cells, GSCs niche, neural stem cells (NSCs), normal glia, tumor-infiltrating lymphocytes, may obscure the leading role of GSCs during gliomagenesis, and make single targeting therapy unsuccessful.

CONCLUSIONSUnderstanding the biological behaviour of GSCs and their regulatory mechanisms may directly impact current efforts for more directed therapeutics against the highly aggressive gliomas. For multiple possible sources to turning into GSCs, simply eradicating the existing GSCs is not enough to be a cure for gliomas, blocking the potential sources of GSCs and ameliorating the local tumor inducing/promoting microenvironment should be a reasonable strategy.

AC133 Antigen ; Antigens, CD ; Biomarkers, Tumor ; metabolism ; Glioma ; metabolism ; pathology ; Glycoproteins ; Humans ; Neoplastic Stem Cells ; metabolism ; pathology ; Peptides

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.
AC133 Antigen ; Antigens, CD ; Antigens, CD34 ; analysis ; Cell Cycle ; Cells, Cultured ; Fetal Blood ; cytology ; Glycoproteins ; analysis ; Hematopoietic Stem Cells ; physiology ; Humans ; Immunophenotyping ; Peptides ; analysis ; Telomerase ; metabolism

The present study was aimed to explore the role of c-Myc gene regulation in maintaining the self-renewal and drug-resistant properties of colon cancer stem cells (CSCs) and the underlying mechanism. CD133(+) cells were isolated by flow cytometry cell sorting from human HT29 cancer cells. A small interfering RNA (siRNA) against c-Myc was used, and the mRNA and protein expressions of c-Myc were investigated by real-time PCR and Western blotting, respectively. To evaluate the effect of c-Myc on the drug resistance of colon CSCs, CD133(+) cells transfected with c-Myc-siRNA were exposed to 5-FU, oxaliplatin, or their combination. The expressions of ATP-binding cassette (ABC) transporters, including ABCG2, ABCB5 and MDR-1, were detected by Western blotting. The results showed that c-Myc was highly expressed in CD133(+) colon CSCs, and the protein and mRNA expressions of c-Myc were effectively blocked by c-Myc siRNA. Furthermore, CD133(+) cells showed significantly increased survival rate in chemotherapy treatment, compared with CD133(-) cells. c-Myc silencing sensitized CD133(+) cells to chemotherapy-induced cytotoxicity and down-regulated the protein expression levels of ABCG2, MDR-1 and ABCB5. These results suggest c-Myc silencing may regulate the expressions of ABC transporters in colon CSCs, and enhance the sensitivity of CSCs to the chemotherapy.
AC133 Antigen ; ATP-Binding Cassette Transporters ; Cell Line, Tumor ; Colon ; Down-Regulation ; Humans ; Neoplastic Stem Cells ; Proto-Oncogene Proteins c-myc ; RNA, Small Interfering

OBJECTIVETo investigate the biology characters of CD133+ cancer stem cells from Hep-2 cell line.

METHODSThe Flow cytometry was applied to purify CD133+ cells from Hep-2 cell line. The sorted CD133+ cells were cultured in RPMI1640. The ability of migration, invasion and clonality of CD133+ cells were performed to characterize the properties of the cells. CD133+ cells and CD133- control cells were treated with paclitaxel and exposed respectively to X-rays emitted by linear accelerator with a dose of 10 Gy. The surviving rates and growth inhibition ratio of cells in two groups were detected with MTT assay to observe the resistance to irradiation and chemotherapy in CD133+ cells.

RESULTSThe percentage of CD133+ cells in the unsorted and the sorted cells were 3.1% +/- 0.2% and 90.2% +/- 5.5%, respectively. CD133+ cells showed the higher proliferation and colony ability than CD133- cells. The numbers of CD133+ and CD133- cells that passed the membrane of Transwell chamber were 526 +/- 39 and 220 +/- 20 respectively (t = 22.08, P < 0.001). The colony forming units of three passages were 30.0% +/- 4.7%, 32.2% + 3.6%, 32.7% + 3.4% in CD133 cells and 15.2% +/- 2.2%, 12.0% + 2.5%, 13.8% +/- 3.3% in CD133- cells. There were statistic difference between two groups (t = 8.99, t = 14.66, t = 12.69, P < 0.01). At 24, 48, and 72 hours of treatment with paclitaxel, the cell surviving rates of CD133+ cells were 90.1% +/- 5.9%, 85.1% +/- 7.1% and 70.3% +/- 6.4% and lower than those of CD133- cells, respectively (t = 5.24, t = 8.18, t = 8.14, P < 0.01) . After radiotherapy, growth inhibition ratio of CD133+ and CD133- cells were 30.0% +/- 7.1% and 55.0% +/- 6.3% (t = 8.30, P < 0.01).

CONCLUSIONSCD133+ cells exist in a small proportion in Hep-2 cell line and they show the properties of cancer stem cells, with the resistance to irradiation and chemotherapy.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Line, Tumor ; Flow Cytometry ; Glycoproteins ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; metabolism ; Neoplastic Stem Cells ; Paclitaxel ; Peptides ; metabolism

OBJECTIVETo detect the expression of CD133 in human larynx tumor cell line, Hep-2 cell line and observe proliferation and differentiation ability of CD133+ groups in vitro.

METHODSImmunocytochemical staining and flow cytometry were used to detect the expression of putative tumor-initiating cell marker CD133 in Hep-2 cell line, and the selective technique of immunomagnetic beads was applied to purify CD133 positive cells. CD133+ tumor cells were cultured and their ability of proliferation and differentiation were observed in vitro.

RESULTSOnly 3. 22% of cells in Hep-2 cell line expressed CD133. In serum-free RPMI1640, On days 3, 5 and 7, their UV absorption were 0. 320,0. 370 and 0. 558 respectively. Compared with CD133 - cells and control Hep-2 cells, CD133 + cells demonstrated increased proliferation capacity. The proportion of CD133+ cells decreased in culture as days passed. In twelve days of culture, the percentage of CD133+ cells decreased from 90. 88% to 4. 53 %.

CONCLUSIONSCD133 was one of makers for tumor-initiating cell of human laryngeal carcinoma, Hep-2 cell line. Its identification would provide a helpful tool to investigate the tumorigenic process of human laryngeal carcinoma and to develop targeted therapies.

AC133 Antigen ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; Carcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Glycoproteins ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; metabolism ; pathology ; Peptides ; metabolism

OBJECTIVETo investigate the expression of CD133 in the bone marrow of patients with myelodysplastic syndrome (MDS) and explore its clinical significance.

METHODSThe expression of CD133 and CD34/CD38 in the bone marrow was detected using flow cytometry in 31 cases of refractory anemia with excess blasts (RAEB), 10 cases of refractory cytopenia with multilineage dysplasia (RCMD) and 11 cases of aplastic anemia (AA).

RESULTSThe percentage of CD133-expressing cells was 6.75% in patients with RAEB, significantly higher than that in patients with RCMD (1.41%) and AA (2.70%) (P<0.05); the percentage of CD133-positive cells were similar between the latter two patient groups (P>0.05). The percentage of CD34(+)/CD38- cells was similar in the 3 groups (P>0.05), all lower than 1%.

CONCLUSIONSAdvanced MDS patients are characterized by an increase of CD133-expressing cells, suggesting the value of CD133 in the diagnosis of RAEB. CD34(+)/CD38- cells do not show a significant value in the diagnosis of MDS.

AC133 Antigen ; Anemia, Aplastic ; metabolism ; Antigens, CD ; metabolism ; Antigens, CD34 ; metabolism ; Female ; Flow Cytometry ; Glycoproteins ; metabolism ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; diagnosis ; metabolism ; Peptides ; metabolism

OBJECTIVETo select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library.

METHODSWith a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique.

RESULTSAfter 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133.

CONCLUSIONWe have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.

AC133 Antigen ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; DNA, Single-Stranded ; Glycoproteins ; metabolism ; Humans ; Neoplastic Stem Cells ; metabolism ; Peptide Library ; Peptides ; metabolism ; Protein Binding ; Sequence Analysis, DNA

OBJECTIVETo construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133(+) SW480 cells in vitro.

METHODSThe two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR. The product obtained was identified by sequencing and inserted into prokaryotic expression vector pEGFP-N1. The product, pEGFP-N1 KNOBδHI, contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop. The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR, then identified by sequencing and inserted into pNEB193, resulting in pNEB-F5. CD133(+) SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope.

RESULTSThe target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR, but not by common PCR. Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133(+) SW480 cells.

CONCLUSIONCompared with common PCR, touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133(+) cancer stem cell-selective adenovirus type 5 vector, which provides carriers for tumor-targeted gene therapy.

AC133 Antigen ; Adenoviridae ; genetics ; Antigens, CD ; Cell Line, Tumor ; Genetic Vectors ; Glycoproteins ; Humans ; Neoplastic Stem Cells ; cytology ; virology ; Peptides ; Plasmids ; genetics ; Polymerase Chain Reaction ; methods