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At least 1,189 records · Page 66Linked to original sources
PubMed2002

The Bmi-1 oncogene induces telomerase activity and immortalizes human mammary epithelial cells.

The vast majority of breast cancers are carcinomas that arise from mammary epithelial cells (MECs). One of the key early events in tumorigenic transformation is the ability of cells to overcome replicative senescence. However, the precise genetic changes that are responsible for this event in MECs is largely unknown. Here, we report that Bmi-1, originally identified as a c-Myc cooperating oncoprotein, can bypass senescence, extend the replicative life span, and immortalize MECs. Furthermore, Bmi-1 was overexpressed in immortal MECs and several breast cancer cell lines. Overexpression of Bmi-1 in MECs led to activation of human telomerase reverse transcriptase (hTERT) transcription and induction of telomerase activity. Telomerase induction by Bmi-1 was an early event in the extension of the replicative life span and immortalization. Bmi-1 was not overexpressed in hTERT-immortalized MECs, suggesting that Bmi-1 functions upstream of hTERT. Although, c-Myc has been reported to induce telomerase in MECs, Bmi-1 appeared to act independently of c-Myc binding sequences in the hTERT promoter. Deletion analysis of the Bmi-1 protein suggested that the RING finger, as well as a conserved helix-turn-helix-turn domain, were required for its ability to induce telomerase and immortalize MECs. These data suggest that Bmi-1 regulates telomerase expression in MECs and plays a role in the development of human breast cancer.

Binding Sites↗
PubMed2004

[Effect of KyoT2 binding protein KBP1 on RBP-J-mediated transcriptional activity].

AIM: To investigate the physical interaction between KyoT2 and KyoT2-binding protein 1 (KBP1) and the effect of KBP1 on RBP-J-mediated transcriptional activity. METHODS: GST-pull down, co-immunoprecipitation, mammalian cell two hybrid assay and reporter gene assay were used to examine the physical and functional interactions between KyoT2 and KBP1. RESULTS: KBP1 and KyoT2 could interact with each other both in-vitro and in-vivo, and overexpression of KBP1 could antagonize the suppressive effect of RING1 on RBP-J. CONCLUSION: KBP1 may indirectly modulate Notch signaling pathway by competing with RING1 for binding sites on KyoT2.

Cell Line↗
PubMed2004

Gene expression changes induced by green tea polyphenol (-)-epigallocatechin-3-gallate in human bronchial epithelial 21BES cells analyzed by DNA microarray.

Many studies suggest green tea is a cancer chemopreventive agent. This effect has been attributed to its major constituent (-)-epigallocatechin-3-gallate (EGCG). EGCG is also observed to have cytotoxic anticancer effects, especially when used in combination with certain chemotherapeutic agents. The biochemical actions of EGCG in chemoprevention and anticancer effects have been studied; however, the mechanisms of action are not clearly understood. We show here by expression genomics the effects of EGCG (25 micromol/L) in the Ha-ras gene transformed human bronchial epithelial 21BES cells. We found induction of temporal changes in gene expression and the coalescence of specific genetic pathways by EGCG. In this experimental system, hydrogen peroxide (H2O2) was produced. By treating cells with EGCG in the presence or absence of catalase, we further distinguished gene expression changes that are mediated by H2O2 from those that are H2O2 independent. Many genes and cellular pathways, including genes of the transforming growth factor-beta signaling pathway, were H2O2 dependent because the effects were abolished by catalase. Gene expression changes that were not affected by catalase included those of the bone morphogenetic protein signaling pathway, peptidylprolyl isomerase (cyclophilin)-like 2, alkylated DNA repair enzyme alkB, polyhomeotic-like 2, and homeobox D1. We show further that EGCG and H2O2 differentially transactivated the bone morphogenetic protein and the transforming growth factor-beta response element promoter reporters, respectively, thus confirming results from DNA microarray analysis. The elucidation of gene expression changes between H2O2-dependent and H2O2-independent responses helps us better understand the cancer chemopreventive and anticancer actions of EGCG.

AlkB Homolog 1, Histone H2a Dioxygenase↗
PubMed2004

[Cancer stem cells in pediatric brain tumors].

Cancers are formed by heterogeneous cell types from immature highly proliferative cells to lineage-committed differentiated cells. Transplantation studies have suggested the existence of "cancer stem cells", individual cells capable of producing an entire tumor. Recent advances in stem cell research have allowed for the demonstration of the existence of cancer stem cells in acute myeloid leukemia, breast cancer, and, most recently, in pediatric brain tumors. Each of these has some similarities with the normal stem cells in the corresponding organs. For example, leukemia stem cells express some, but not all, markers of hematopoietic stem cells. Regarding pediatric brain tumors, putative cancer stem cells were identified from medulloblastoma and also from glioma. These tumor-derived cells self-renew under clonal conditions, and differentiate into neurons and glia as well as into abnormal cells with mixed phenotypes. Interestingly, the tumor stem/progenitors, enriched in culture, maintained proliferation after 4 weeks from transplantation into neonatal rat brain. In this review, we discuss the difference as well as the similarity between tumor and normal stem cells, and also the possible clinical implication of cancer stem cells.

Animals↗
PubMed2005

Hematopoietic stem cells, leukemic stem cells and chronic myelogenous leukemia.

Blood-related cancers, or leukemias, have been shown to arise from a rare subset of cells that escape normal regulation and drive the formation and growth of the tumor. The finding that these so-called cancer stem cells, or leukemic stem cells (LSC), can be purified away from the other cells in the tumor allows their precise analysis to identify candidate molecules and regulatory pathways that play a role in progression, maintenance, and spreading of leukemias. The analyses of the other, numerically dominant, cells in the tumor, while also interesting, do not directly interrogate these key properties of malignancies. Mouse models of human myeloproliferative disorder and acute myelogenous leukemia have highlighted the remarkable conservation of disease mechanisms between both species. They can now be used to identify the LSC for each type of human leukemia and understand how they escape normal regulation and become malignant. Given the clinical importance of LSC identification, the insights gained through these approaches will quickly translate into clinical applications and lead to improved treatments for human leukemias.

Animals↗
PubMed2005

[Differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells].

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.

Cell Differentiation↗
PubMed2005

Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation.

BACKGROUND: Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression. METHODS: A transcriptional repressor, Bmi-1 cDNA was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) of its mRNA from K562 cells. A plasmid expressing antisense Bmi-1 mRNA was then constructed by reverse design of PCR primers and cloned to the plasmid pLNCX2; G418 was added to the medium after the plasmid was successfully introduced in K562 cells by lipofectin-mediated DNA transfection. The effects of the antisense expression on the proliferation of K562 cells were analyzed by using microculture tetrazolium and colony forming. Cell cycle was analyzed by using flow cytometry. The p16 expression of K562 cells was observed by immunofluorescence histochemical stain. RESULTS: K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P < 0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls. CONCLUSION: The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

Cell Cycle↗
PubMed2005

[Construction of eukaryotic expression vector for HPC2 and its expression in HEK293 cells].

OBJECTIVE: To construct the eukaryotic expression vector for HPC2 for expression in HEK293 cells. METHODS: HPC2 from pcDNA3/HPC2 were inserted into the flag-tagged vector pcDNA3-flag by subcloning method. The recombinant plasmid pcDNA3-flag/HPC2 was then transfected into HEK293 cells using a routine lipofectamine method. The cell lysate was used for Western blotting to examine the expression of the target protein. RESULTS AND CONCLUSION: Double restriction enzyme digestion and DNA sequencing indicated successful construction of the eukaryotic expression vector for HPC2 and the fusion protein was highly expressed in HEK293 cells, which provides an important basis for functional study of HPC2.

Cell Line↗
PubMed2005

[Inhibition effect of antisense Bmi-1 on Jurkat cells].

OBJECTIVES: To investigate whether antisense Bmi-1 plasmid could inhibit the proliferation of Jurkat cells. METHODS: The antisense plasmid was constructed by PCR amplification of a 171 bp segment spanning Bmi-1 start codon and zinc finger structure and the PCR product was subsequently inserted reversely to plasmid pLNCX2. The final construct was confirmed through restriction enzyme digestion. G418 was added into the medium after the plasmid was successfully introduced into Jurkat cells by using lipofectin-mediated DNA transfection. The proliferation of Jurkat cells were determined by MTT and colony formation assays. Cell cycle was determined by flow cytometry. The p16 expression of Jurkat cells was studied by immunofluorescent histochemistry. RESULTS: The growth rate of antisense Bmi-1 transfected Jurkat cells was significantly lower than that of the controls, and the colony forming capacity of the transfected cells decreased significantly (P < 0.01), the colony numbers being (90.7 +/- 9.07)/10(3) cells, (83.3 +/- 6.11)/10(3) cells and (56.0 +/- 5.56)/10(3) cells for control cells, empty plasmid transfected Jurkat cells and antisense Bmi-1 transfected Jurkat cells, respectively. The percentage of G, phase cells was increased and the p16 expression of antisense Bmi-1 transfected cells was significantly upregulated than that of control cells. CONCLUSION: Antisense Bmi-1 can inhibit the growth and upregulate the expression of p16 of Jurkat cells in vitro.

Cell Cycle↗
PubMed2006

[Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21].

OBJECTIVE: To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21. METHODS: Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot. RESULTS: Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody. CONCLUSION: There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.

Animals↗
PubMed2006

Histological type of oncogenity and expression of cell cycle genes in tumor cells from human mesenchymal stem cells.

In previous experiments, a novel tumor cell line, which was characterized by dominated F6 mutated from human mesenchymal stem cells (hMSCs), was developed. The mechanism and biological characteristics of this mutation are still unclear. In this study, the histological type of F6 cells was investigated by immunohistochemistry with specific markers: vimentin, CD117, desmin, NSE and vWF. The characteristics of proliferation and metastasis were shown by PCNA (proliferating cell nuclear antigen), and nm23 and cell cycle-related genes, such as p16, p21, p53 and pRb, were analyzed by RT-PCR and immunohistochemistry. The expression of hTRAP and BMI-1 were detected by real-time PCR and Western blotting. The activity of telomerase was analyzed by TRAP (telomerase repeat amplification protocol) assay. The results showed that multi-directional differentiation occurred in F6 cells, i.e., special markers of muscle, endothelial cell and nerve system were co-expressed in F6 cells, while hardly expressed in hMSCs. F6 cells maintained the same properties as of MSCs, such as negativity for both CD117 and vimentin. F6 cells exhibited strong positivity for PCNA and negativity for nm23. The cell cycle-related genes, such as p16, p21, p53 and pRb, were not detected in F6 cells, while the expression of hTRAP and BMI-1 was significantly higher. The activity of telomerase was also significantly higher in F6 cells than that in hMSCs. These findings indicated that multi-directional differentiation occurred during the transformation of hMSCs into F6 cells, and that the genes of cell cycle and cell senescence may also be associated with the neoplasia of adult stem cells.

Acid Phosphatase↗
PubMed2006

RNF2 interacts with the linker region of the human P-glycoprotein.

The human P-glycoprotein (Pgp) is a drug-efflux pump responsible for innate or acquired multidrug resistance in many cancers. Pgp contains a unique approximately 75 amino acid long linker region in its middle, which is critically important for its drug transport and ATPase functions. To identify cellular proteins that bind to this linker region and modulate Pgp function, a yeast two-hybrid analysis was carried out. This procedure identified RNF2 (RING finger protein 2), an E3 ubiquitin ligase, as a prominent Pgp-interacting protein. Co-expression of RNF2 with Pgp in Sf9 insect cells resulted in decreased ATPase activity and proteolytic protection of the transporter protein. Immunoprecipitation experiments confirmed the physical interaction between these two proteins. Confocal microscopy showed the presence of RNF2 in the cytoplasm of the Pgp-negative, drug-sensitive MCF-7 breast cancer cells. However, it was undetectable in the Pgp-positive and drug-resistant MCF-7 cells. We suggest that RNF2 regulates the cellular abundance of Pgp, and plays a key role in the development of cancer drug resistance through its own down-regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Expression of novel DNA-binding protein with zinc finger structure in various tumor cells.

We have isolated from B16 mouse melanoma cells a complementary DNA (Mel-18), whose deduced amino acid sequence possesses a characteristic zinc finger structure. Immunostaining with antibodies raised against partial Mel-18 peptide sequences demonstrated nuclear localization of the gene product. We have also demonstrated that this protein has DNA-binding capacity, and the zinc finger is responsible for the DNA binding. At the transcriptional level the Mel-18 mRNA was detected in all tumor cells examined as well as melanoma cells (ontogenically of neural origin) but was scarcely present in normal tissues except neural organs. The transcript is developmentally regulated. These data suggest that Mel-18 may play a role in transcriptional regulation and also in control of cell proliferation and/or neural cell development.

Amino Acid Sequence↗