Journal Article

Down-Regulation of DNA Mismatch Repair Enhances Initiation and Growth of Neuroblastoma and Brain Tumour Multicellular Spheroids

Collins, Samuel L., Hervé, Rodolphe, Webb, Jeremy S., Keevil, C. W., Blaydes, Jeremy P.

2011

Overview

Multicellular tumour spheroid (MCTS) cultures are excellent model systems for simulating the development and microenvironmental conditions of in vivo tumour growth. Many documented cell lines can generate differentiated MCTS when cultured in suspension or in a non-adhesive environment. While physiological and biochemical properties of MCTS have been extensively characterized, insight into the events and conditions responsible for initiation of these structures is lacking. MCTS are formed by only a small subpopulation of cells during surface-associated growth but the processes responsible for this differentiation are poorly understood and have not been previously studied experimentally. Analysis of gene expression within spheroids has provided clues but to date it is not known if the observed differences are a cause or consequence of MCTS growth. One mechanism linked to tumourigenesis in a number of cancers is genetic instability arising from impaired DNA mismatch repair (MMR). This study aimed to determine the role of MMR in MCTS initiation and development. Using surface-associated N2a and CHLA-02-ATRT culture systems we have investigated the impact of impaired MMR on MCTS growth. Analysis of the DNA MMR genes MLH1 and PMS2 revealed both to be significantly down-regulated at the mRNA level compared with non-spheroid-forming cells. By using small interfering RNA (siRNA) against these genes we show that silencing of MLH1 and PMS2 enhances both MCTS initiation and subsequent expansion. This effect was prolonged over several passages following siRNA transfection. Down-regulation of DNA MMR can contribute to tumour initiation and progression in N2a and CHLA-02-ATRT MCTS models. Studies of surface-associated MCTS differentiation may have broader applications in studying events in the initiation of cancer foci.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Adaptor Proteins, Signal Transducing - antagonists & inhibitors / Adaptor Proteins, Signal Transducing - genetics / Adenosine Triphosphatases - antagonists & inhibitors / Adenosine Triphosphatases - genetics / Animals / Biology / Brain / Brain cancer / Brain Neoplasms - genetics / Brain Neoplasms - pathology / Brain research / Brain tumors / Cancer / Cancer research / Cell culture / Computer simulation / Deoxyribonucleic acid / Differentiation / DNA / DNA Mismatch Repair - genetics / DNA repair / DNA Repair Enzymes - antagonists & inhibitors / DNA Repair Enzymes - genetics / DNA-Binding Proteins - antagonists & inhibitors / DNA-Binding Proteins - genetics / Down-Regulation / Gene expression / Gene Expression Regulation, Neoplastic / Gene silencing / Genes / Genomic instability / Growth / Humans / Hypoxia / Ionizing radiation / Leukemia / Medical research / Medicine / Melanoma / Metastasis / Mice / Mismatch repair / Mismatch Repair Endonuclease PMS2 / MLH1 protein / Mutagenesis / Mutation / MutL Protein Homolog 1 / Neuroblastoma / Neuroblastoma - genetics / Neuroblastoma - pathology / Nuclear Proteins - antagonists & inhibitors / Nuclear Proteins - genetics / Physiological aspects / Physiology / Real-Time Polymerase Chain Reaction / Repair / Reverse Transcriptase Polymerase Chain Reaction / Ribonucleic acid / RNA / RNA, Messenger - genetics / RNA, Small Interfering - genetics / siRNA / Spheroids / Spheroids, Cellular - pathology / Stability / Transfection / Tumor cell lines / Tumor Cells, Cultured / Tumorigenesis / Tumors