Cancer stem cells are defined as rare cells in cancer tissues with indefinite potential for self-renewal that drives tumorigenesis. It was first extensively documented for leukaemia and multiple myeloma. It has also been found in solid cancers such as human breast cancer and nervous system tumors. Studies of cancer stem cell biology and mechanisms of tumorigenesis are lending insight into the origins of cancer and will ultimately yield new approaches to fight cancer.
Animals ; Cell Transformation, Neoplastic ; Humans ; Mice ; Neoplasms ; pathology ; therapy ; Neoplastic Stem Cells ; cytology ; Tumor Stem Cell Assay ; methods

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.
Adolescent ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; immunology ; metabolism ; Male ; Neoplasm, Residual ; immunology ; metabolism ; Neoplastic Stem Cells ; cytology ; Tumor Stem Cell Assay

OBJECTIVETo explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene mutation induced by ethyluitrosourea (ENU).

METHODSSingle cell cloning culture, two-way screening, multiple PCR amplification and electrophoresis technique were used.

RESULTSWith dose of ENU increasing, cell plating efficiency reduced (in the group with 100-200 micro g/ml doses, P < 0.01) and mutation frequency increased (in the group with 12.5-200.0 micro g/ml doses, P < 0.05) significantly. There was no all exons deletion in spontaneous mutations, and only 7.7% of them were detected as single exon deletion. But, deletion was found in 79.7% of ENU-induced mutations (62.5%-89.4%, P < 0.01), and deletion mutations in all nine exons of HPRT gene. Most of ENU-induced mutations were chain deletion with multiple exons (88.1%).

CONCLUSIONSThe spectra in spontaneous mutations differed completely from ENU-induced ones. ENU was liable to cause bigger changes in genetic structure, which suggested a stronger ENU's mutagenesis.

Alkylating Agents ; pharmacology ; Ethylnitrosourea ; pharmacology ; HL-60 Cells ; Humans ; Hypoxanthine Phosphoribosyltransferase ; genetics ; metabolism ; Leukemia, Myeloid ; pathology ; Mutation ; drug effects ; Tumor Stem Cell Assay

A new interactive image segmentation method used in the multiple myeloma cloning spots image segmentation is presented in the paper. Based on the theory of graph cuts, some pixels are selected as the front object and the background seeds, and the other parts are treated as the unknown region. Then, an energy function is constructed and initialized through K-means, and the minimum cut method is used in the segmentation by energy minimization. Last, the image is eroded and dilated, and the cloning separate parts could be got effectively. For the pixels which may be partitioned wrongly, we use a tool similar to a brush to re-mark the front object or the background, and divide once again. Both subjective the evaluation criteria and the RUMA, evaluation criteria are used to evaluate the method, and the experiment results are satisfactory.
Humans ; Image Enhancement ; methods ; Image Interpretation, Computer-Assisted ; methods ; Multiple Myeloma ; classification ; Pattern Recognition, Automated ; methods ; Tumor Stem Cell Assay ; instrumentation ; methods

AIMTo study the effect of the combined use of genistein and ionizing radiation (IR) on prostate DU145 cancer cells.

METHODSDU145, an androgen-independent human prostate cancer cell line, was used in the experiment. Clonogenic assay was used to compare the survival of DU145 cells after treatments with genistein alone and in combination with graded IR. Apoptosis was assayed by DNA ladder and TUNEL stain. Cell cycle alterations were observed by flow cytometry and related protein expressions by immunoblotting.

RESULTSClonogenic assay demonstrated that genistein, even at low to medium concentrations, enhanced the radiosensitivity of DU145 cells. Twenty-four hours after treatment with IR and/or genistein, apoptosis was mainly seen with genistein at high concentrations and was minimally related to IR. At 72 h, apoptosis also occurred in treatment with lower concentration of genistein, especially when combined with IR. While both IR and genistein led to G2/M cell cycle arrest, combination of them further increased the DU145 cells at G2/M phase. This G2/M arrest was largely maintained at 72 h, accompanied by increasing apoptosis and hyperdiploid cell population. Cell-cycle related protein analysis disclosed biphasic changes in cyclin B1 and less dramatically cdc-2, but stably elevated p21 cip1 levels with increasing genistein concentrations.

CONCLUSIONGenistein enhanced the radiosensitivity of DU145 prostate cancer cells. The mechanisms might be involved in the increased apoptosis, prolonged cell cycle arrest and impaired damage repair.

Androgens ; physiology ; Anticarcinogenic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA, Neoplasm ; analysis ; biosynthesis ; genetics ; Flow Cytometry ; Genistein ; pharmacology ; Humans ; Immunoblotting ; In Situ Nick-End Labeling ; Male ; Prostatic Neoplasms ; drug therapy ; radiotherapy ; Tumor Stem Cell Assay

To investigate the effect of manganese superoxide dismutase (MnSOD) silence on the in vitro tumorigenicity in human small cell lung cancer NCI-H446 cells and the underlying mechanisms.
 Methods: Sphere formation cells from NCI-H446 cells were obtained by suspension culture, while the expression of MnSOD and urokinase type plasminogen activator (uPAR) was analyzed by Western blot. Silence of MnSOD was performed by adenovirus infection in the second passage formation cells, and the effect of MnSOD silence on tumorigenicity in NCI-H446 cells was evaluated by sphere formation assay and soft-agar colony formation assay, while the expression of uPAR was analyzed by Western blot.
 Results: Compared with NCI-H446 cells, the sphere formation rate, colony formation rate, and the expression of MnSOD and uPAR were significantly increased in the second passage sphere formation cells in NCI-H446 cells (P<0.05). Silence of MnSOD inhibited the sphere formation rate, colony formation rate, and the expression level of uPAR in the second passage sphere formation cells in NCI-H446 cells.
 Conclusion: MnSOD may promote tumorigenicity in NCI-H446 cells by up-regulation of uPAR expression in vitro.
Adenoviridae ; Carcinogenesis ; Cell Line, Tumor ; Humans ; In Vitro Techniques ; Lung Neoplasms ; etiology ; metabolism ; RNA Interference ; Receptors, Urokinase Plasminogen Activator ; genetics ; metabolism ; Small Cell Lung Carcinoma ; etiology ; metabolism ; Spheroids, Cellular ; pathology ; Superoxide Dismutase ; genetics ; metabolism ; Tumor Stem Cell Assay ; Up-Regulation

Though the malignancy of a tumor is generally postulated to be affected by the degree of differentiation of tumor cells, the relationship between differentiation and malignancy of tumors has not been clearly elucidated. Using in vitro established mouse(B16) and human(IGR3) melanoma cell lines, we performed various in vitro and in vivo experiments to clarify the relationship between melanogenicity and malignancy. High and low melanin-producing clones were selected from both cell lines by the limiting dilution technique and their melanogenicities were confirmed by the determination of melanin quantity and tyrosinase activity along with electron microscopic examination. Selected clones from both cell lines revealed that low melanin-producing clones showed a slightly broader chromosomal distribution, a shorter doubling time with a higher DNA synthesis and a greater colony forming capacity in semi-solid agar medium than those of high melanin-producing clones. The low melanin-producing clone derived from B16 also had a lower tumor-take dose and a more rapid tumor-growth rate than the high melanin-producing counterpart following transplantation into syngeneic mice. These results support the concept that the melanogenicity and other biological characteristics associated with malignancy are inversely related in malignant melanoma cells.
Animal ; Cell Line ; Human ; Melanins/*metabolism ; Melanoma/*pathology ; Melanoma, Experimental/*pathology ; Mice ; Mice, Inbred Strains ; Microscopy, Electron ; Neoplasm Transplantation ; Skin Neoplasms/*pathology ; Tumor Cells, Cultured/*pathology ; Tumor Stem Cell Assay

OBJECTIVE: To investigate the effect of Bcl-2 antisense oligodeoxynucleotides (ASODN) on the cell proliferation and Bcl-2 expression in bile duct carcinoma cell line QBC939. METHODS: Bcl-2 ASODN and control sequence were transfected into cell line QBC939 by Lipofectamine 2000. The changes of Bcl-2 protein were detected by Western blot. The survival rate and colony formation rate of QBC939 cells incubating with Bcl-2 ASODN were evaluated by trypan blue staining assay and colony forming test. RESULTS: The densitometric analysis of Gel-photograph showed that the level of Bcl-2 protein expression in the ASODN transfected group was significantly lower than that in the controls (P < 0.01). Both trypan blue staining assay and colony forming test demonstrated that Bcl-2 ASODN could partially inhibit the growth of QBC939 cells. After incubating with Bcl-2 ASODN, the survival rate and colony formation rate of QBC939 cells were significantly lower than those of the controls (P < 0.05). CONCLUSION Bcl-2 ASODN inhibits the cell proliferation in bile duct carcinoma cell line QBC939 by blocking the expression of bcl-2 gene.
Bile Duct Neoplasms ; metabolism ; pathology ; Cell Division ; drug effects ; Humans ; Lipids ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Tumor Stem Cell Assay

BACKGROUND: Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of cord blood (CB) hematopoietic stem cells for transplantation. As well as stem cell number, stromal cells are necessary for functional maturation of hematopoiesis. The purpose of this study was to analyze the development of stromal cells during ex vivo expansion of CB CD34+ cells. METHODS: CD34+ cells were purified from CB by magnetic bead selection. The levels of of interleukin-3, interleukin-1 beta, interleukin-6, granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha were measured in culture supernatants on 0, 1, 2, and 3 weeks, using ELISA techniques. CB CD34+ cells were expanded in Iscoves modified Dulbeccos medium in the presence of several cytokines. The expression of E-selectin, vascular cell adhesion molecule- 1, intercellular adhesion molecule- 1, platelet/endothelial cell adhesion molecule-1, von Willebrand factor, vimentin, and CD14 in newly developed stromal cells was examined by immunocytochemical method. Relevant extracellular matrix (ECM) proteins and proper cytokines were also assayed for the most suitable condition for expansion of stromal cells. RESULTS: Several cytokines were found to have been produced by CB CD34+ cells as well as bone marrow-derived CD34+ cells. During ex vivo expansion of CB CD34+ cells, stromal cells appeared in the culture by day 4 and expanded over the following 7- 10 days before being confluent by day 2 1. These cells expressed surface markers characteristic of cells of endothelial lineage. Furthermore, these stroaml cells also expanded effectively when treated with thrombopoietin+flt-3 ligand+stem cell factor+leukemia inhibitory factor or 0. 1% poly-L-lysine-coated wells. CONCLUSION: Stromal cells were developed during ex vivo expansion of CB CD34+ cells and that this development could be enhanced further by treating the stromal cells with cytokines or ECM.
Cell Adhesion ; Cytokines ; E-Selectin ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; Fetal Blood* ; Granulocytes ; Hematopoiesis ; Hematopoietic Stem Cells ; Interleukin-1beta ; Interleukin-3 ; Interleukin-6 ; Stem Cells ; Stromal Cells* ; Tumor Necrosis Factor-alpha ; Vimentin ; von Willebrand Factor

The discovery of induced pluripotent stem cells(iPSCs) is a promising advancement in the field of regenerative medicine. Previous studies have indicated that the teratoma-forming propensity of iPSCs is variable; however, the relationship between tumorigenic potential and genomic instability in human iPSCs (HiPSCs) remains to be fully elucidated. Here, we evaluated the malignant potential of HiPSCs by using both colony formation assays and tumorigenicity tests. We demonstrated that HiPSCs formed tumorigenic colonies when grown in cancer cell culture medium and produced malignancies in immunodeficient mice. Furthermore, we analyzed genomic instability in HiPSCs using whole-genome copy number variation analysis and determined that the extent of genomic instability was related with both the cells' propensity to form colonies and their potential for tumorigenesis. These findings indicate a risk for potential malignancy of HiPSCs derived from genomic instability and suggest that quality control tests, including comprehensive tumorigenicity assays and genomic integrity validation, should be rigorously executed before the clinical application of HiPSCs. In addition, HiPSCs should be generated through the use of combined factors or other approaches that decrease the likelihood of genomic instability.
Animals ; Carcinogenesis ; Cells, Cultured ; DNA Copy Number Variations ; Genomic Instability ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; transplantation ; Mice ; Mice, SCID ; NIH 3T3 Cells ; Octamer Transcription Factor-3 ; metabolism ; Teratocarcinoma ; etiology ; Teratoma ; etiology ; Tumor Stem Cell Assay