Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells
Introduction
In the design of biological processes for the production of therapeutic and/or diagnostic compounds from mammalian cells, it is vital to take into account the properties of the cell line being used (Chu et al., 2005). Details pertaining to a cell line in terms of its: growth pattern, nutritional requirements, glycosylation capabilities, and response to stimuli are important parameters to consider in order to properly design specific processes (Lum et al., 2004).
An important cellular property in biotechnology applications is adhesion—a cell's ability to attach to a surface in order to grow (Zhu et al., 2002). Depending on the exact application, a cell line that does not adhere to a surface, anchorage-independent, may be strongly preferred over a cell line that adheres to a surface, anchorage-dependent (Lum et al., 2004; Karu et al., 2001). For a different application, an anchorage-dependent cell line may be preferable over an anchorage-independent cell line. Being able to manipulate the cellular feature of adhesion would, therefore, benefit biotechnology applications and is the basis of the current study (Lum et al., 2004; Zhu et al., 2002).
A variety of studies have been conducted to evaluate the importance of cellular properties for the production of specific products. Researchers have also identified possible pathways to modify cellular properties by employing specific selection methods. In relation to adhesion, most studies have focused on either quantifying observations on a genetic level or exploring the effects of specific compounds (Springer et al., 1976). For instance, selenite, a hydrous calcium sulfate, has been shown to reduce the ability of HeLa cells to attach to fibronectin (Zhu et al., 1998). Researchers have also shown that blocking the expression of pten, a tumor suppressor gene, in 293T cells using siRNA resulted in a loss of adhesion as well as a change in morphology (Mise-Omata et al., 2005; Crowther, 2002). Additional studies have highlighted a number of genes thought to be involved in mediating adhesion such as rhoA, rac1, and cdc42; although the exact mechanisms have not been fully elucidated (Mise-Omata et al., 2005; Hatzimanikatis and Lee, 1999; Tavazoie et al., 1999).
In the present study DNA microarrays were used to identify genomic differences between anchorage-independent and anchorage-dependent HeLa cells. Data generated from these arrays were normalized and then screened using a variety of imaging tools. Based on the results of several clustering algorithms, a review of previous studies, and their relative expression levels, two genes were selected for further investigation. The expression levels of these two genes, siat7e and lama4, were verified using reverse transcription-polymerase chain reaction (RT-PCR). Once verified, the expression level of either gene was manipulated and the adhesion features of these ‘altered’ cells were characterized first using a particle counter and then a shear flow chamber.
Section snippets
Cell culture growth and sampling
The two cell lines, anchorage-dependent and anchorage-independent HeLa cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) (Catalog Nos. CCL-2 and CCL-2.2, respectively). Both cell lines were grown in BioFlo 3000 bioreactors (New Brunswick Scientific Co., Edison, NJ, USA) with a working volume of 1.5 L. Runs were conducted for up to 7 days after inoculation with constant sampling to characterize growth parameters. At least two different runs were carried out
Growth of anchorage-dependent and anchorage-independent HeLa cells
Growth characterization of anchorage-dependent and anchorage-independent HeLa cells was performed in bioreactors under the same set of conditions. Samples for microarray analysis were taken at the same time point, when both cell lines were in exponential growth. Both cell lines were experiencing similar environments as determined by measuring metabolic concentrations, cell density, pH, and viability. For example, both cell lines had a glucose concentration of approximately 3 g/L and a lactate
Discussion
In the present study, DNA microarrays along with other genomics tools were used to identify the genes siat7e and lama4 as influential to the adhesion of HeLa cells. The selection of these two genes from a broader list of differentially expressed genes was based on: proposed or known functionalities detailed in previous studies, expression levels, and clustering outcomes. Anchorage-independent HeLa cells exhibited higher expression of siat7e and lower expression of lama4 relative to
Acknowledgments
Funding was provided by the Intramural program at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. The authors would like to thank Renee Rubio and Dr. John Quackenbush, formerly at TIGR for their assistance with the cDNA microarrays. The authors would also like to thank Susan Napier at Johns Hopkins University for her help and expertise with the shear flow chamber experiments.
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