Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells

https://doi.org/10.1016/j.ymben.2006.12.001Get rights and content

Abstract

The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4.

Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin α4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion.

Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives.

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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