RESUMO
Epithelial-to-mesenchymal transition (EMT) as well as the reverse process, mesenchymal-to-epithelial transition (MET) is important during embryogenesis. EMT is also involved in cancer invasion and metastasis, and can generate cells with properties similar to those of stem cells. Retrotransposons can rearrange the genome by inserting DNA in new loci, thus inducing mutations. This study examines the gene expression of transcription factors involved in EMT and MET. In the second experimental panel, the gene expression of L1 retrotransposon was studied. L1-open reading frame (ORF) 2 mRNA was found to be expressed both in cancer and cancer stem cells, while L1-ORF1 mRNA was expressed only in cancer cells. The suppression of L1-ORF2 gene expression demonstrated that this retrotransposon might affect EMT in colon cancer stem cells. This study highlights that the EMT process seems to differ between cancer cells and cancer stem cells, and that transposable elements seem to be involved in the process, influencing cellular plasticity.
Assuntos
Plasticidade Celular/fisiologia , Endonucleases/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Endonucleases/antagonistas & inibidores , Endonucleases/genética , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homeobox Nanog , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fases de Leitura Aberta/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , DNA Polimerase Dirigida por RNA/genética , Ribonucleoproteínas/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The Metnase fusion gene consists of a SET histone methyltransferase domain and a transposase domain from Mariner transposase. This transposable element is involved in chromosome decatenation, enhances DNA repair, promotes foreign DNA integration, and assists topoisomerase II function. This study investigates the role of Metnase in colon cancer homeostasis and maintenance of the stemness phenotype in colon cancer stem cells (CSCs). Silencing of Metnase was performed in human cancer cell lines before and after treatment with cisplatin, and in colon CSCs. Subsequent changes in the expression of genes involved in repair mechanisms, DNA synthesis, topoisomerase II function, and metastasis as well stemness transcription factors were studied with RT-qPCR experiments. Cellular viability and apoptosis were evaluated by flow cytometry. The results suggest that Metnase influences the expression of many genes involved in the above processes. Furthermore, Metnase levels appear to impact upon expression of NANOG, OCT3/4, and SOX2. Suppression of Metnase also led to an increase in apoptosis. Therefore, Metnase may possess an important role in DNA repair, topoisomerase II function, and the maintenance of stemness during colon cancer development.
Assuntos
Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas de Fusão Oncogênica/genética , Transposases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Neoplasias do Colo/patologia , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Células-Tronco NeoplásicasRESUMO
BACKGROUND: Determination of response to chemotherapy is a major requirement of personalized medicine. Resistance, whether developed or native, critically affects a treatment's success. Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer. RESULTS: We treated human breast and colon cancer cell lines with melphalan, cisplatin, mechlorethamine or doxorubicin, as monotherapies. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types. CONCLUSION: The comet assay technique can provide reliable and quick results with minimum requirements and is applicable to a wide variety of drugs.
