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Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment.

ABSTRACT: BACKGROUND: There is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells under conditioned culture. Furthermore the gene regulation pathway involved in cellular reprogramming process was investigated. METHODS: The selection and identification of SP in 50 CNE-2 single cell clones were performed by flow cytometry. The transwell assay and immunofluorescence staining were used to measure migration and cancer stem cell characters of non-SP single clone cells cultured with conditioned medium respectively. The subtraction suppression hybridization (SSH) technique and northern blotting analysis was applied to explore the pluripotency-associated genes under a unique epigenetic sub-microenvironment. RESULTS: Among 50 clones, only one did not possess SP subpopulation while others did. The non-SP cells induced by macrophage-like cells showed more aggressive characters, which increased cell migration compared with the control cells and showed some fraction of SP phenotype. These cells expressed distinguished level of pluripotency-associated genes such as ADP-ribosylation factor-like 6 interacting protein (ARMER), poly (rC) binding protein 1 (PCBP1) and pyruvate dehydrogenase E1-beta subunit (PDHB) when subjected to the environment. CONCLUSION: To our knowledge, this is the first study to demonstrate that non-SP single-clone cells can be induced to generate a SP phenotype when they are cultured with conditioned medium of macrophage-like cells, which is associated with the reactivation of pluripotency-associated genes.

SUBMITTER: Cao JX

PROVIDER: S-EPMC2837014 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment.

Cao Jun-Xia JX Cui Yu-Xin YX Long Zi-Jie ZJ Dai Zhong-Min ZM Lin Ji-Yan JY Liang Yi Y Zheng Fei-Meng FM Zeng Yi-Xin YX Liu Quentin Q

BMC cancer 20100225

<h4>Background</h4>There is increasing evidence that cancers contain their own stem-like cells, and particular attention has been paid to one subset of cancer-stem cells termed side population (SP). Stem cells under normal physical conditions are tightly controlled by their microenvironment, however, the regulatory role of the microenvironment surrounding cancer stem cells is not well characterized yet. In this study we found that the phenotype of SP can be "generated" by macrophage-like cells u ...[more]

PMID: 20181293

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Project description:Nasopharyngeal carcinoma (NPC) is a common malignant tumor. In recent studies, we demonstrated that overexpression of the Bax inhibitor-1 (BI-1) induces cell transformation in NIH3T3 cells and that knockdown of BI-1 and human telomerase reverse transcriptase (hTERT) gene expression suppresses NPC cell proliferation and induces apoptosis. To evaluate the combination anti-tumor effects of siRNAs against hTERT and BI-1 in the CNE-2 NPC cell line, combined and separate short-hairpin (sh)RNA plasmids targeting hTERT and BI-1, respectively, were constructed. hTERT and BI-1 mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation, colony formation and migration ability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), soft agar and wound healing assay. Cell apoptosis was observed by flow cytometry, Hoechst 33258 staining and caspase-3 activity. hTERT, BI-1 and combined shRNA plasmids were injected into xenograft NPC tumor tissues, and expression of hTERT and BI-1 was detected by real-time PCR and immunohistochemistry. Tumor growth was measured by tumor volume and apoptosis in vivo was confirmed by TdT-mediated dUTP nick end-labeling (TUNEL). Our results showed that combined shRNA specific for hTERT and BI-1 markedly suppressed hTERT and BI-1 gene expression in vitro and in vivo. In addition, CNE-2 cell proliferation was inhibited in vitro as well as in vivo. Following the knockdown of the two gene expressions, CNE-2 exhibited a decrease in colony formation and migration ability and an increase in the apoptotic rate compared to the control groups. Our in vitro and in vivo study showed that the combinative silencing of the two genes enhanced the therapeutic effect compared to the silencing of each individual shRNA. These data suggested that combinatorial gene therapy targeting hTERT and BI-1 may be beneficial as a tumor therapy strategy against human NPCs.

Dataset Information

Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment.

Publications

Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment.

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