Abstract
Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.
Highlights
Microgravity on space flights has been shown to affect the physiology of a cell considerably [1]
Cell lines were procured from the National centre for cell science, Pune, India and were maintained in DMEM-F12 (DLD-1) or RPMI1640 (MOLT-4, HL-60) medium supplemented with 10% fetal bovine serum (Life Technologies, USA) at 37°C in a humidified 5% CO2 incubator in 25mm3 tissue culture plates (TCP) and in 10ml3 high aspect ratio vessels (HARV) within a rotating cell culture system (RCCS)
It is of significance that cancer cell proliferation and progression can be altered by microgravity in DLD-1 cells and to a lesser extent, in MOLT-4 cells, as demonstrated by this study
Summary
Microgravity on space flights has been shown to affect the physiology of a cell considerably [1]. The weightlessness and reduced acceleration (less than 1 g) in space, removes the effect of gravity, allowing cell cultures in space to have unhindered movement of the culture medium, a shear free environment and, as cells are not bound by any directional force, unrestricted movement of cells within the medium Under such conditions cells tend to coalesce and form aggregates creating three dimensional (3D) environments where they interact on multiple planes [2]. The RCCS is a mechanical system that simulates reduced gravity on earth by canceling the directional vector through constant rotation of a High Aspect Ratio Vessel (HARV) This maintains cells in a constant free fall and a shear free environment allowing cells to coalesce and form 3D aggregates [2]. The gene expression profile revealed dysregulation of microRNA host genes in microgravity including the significant tumor suppressor, miR-22 in DLD-1
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