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

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, anchoragedependent 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 anchorageindependent 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.

Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

“…In a previous study, we identified two genes that have a role in cell adhesion (20): siat7e, a type II membrane glycosylating sialyltransferase, and lama4 which encodes laminin ␣4, a member of the laminin family of glycoproteins. These two genes were identified following a comparison of gene transcription of two phenotypically distinct HeLa cells, anchorage-dependent and anchorageindependent.…”

“…In a previous study we compared the transcription profiles of anchorage-dependent and anchorage-independent HeLa cells using DNA microarrays (20). The gene siat7e (ST6GalNac V) was identified as one of the genes that play a role in controlling the degree of cell adhesion.…”

“…The gene siat7e (ST6GalNac V) was identified as one of the genes that play a role in controlling the degree of cell adhesion. It was shown that higher siat7e transcription corresponded to a lower degree of adhesion by microscopic evaluation and by monitoring cell detachment in a shear flow chamber (20), and inhibiting siat7e transcription using siRNA was followed by enhanced adhesion. The human sialyltransferase ST6GalNac V, a member of the ST6GalNac family of sialyltransferases, is a type II Golgi membrane protein that transfers sialic acid from the donor CMP-Neu5Ac to the GalNac residue on the ganglioside, GM1b, forming GD1␣.…”

Mammalian cells as biopharmaceutical production hosts in the age of omics

“…Approximately 1000 protein spots were detected on each gel. Spots, which were identified as being differentially expressed throughout culture and bright enough to allow for further processing (66), were analyzed using LC-MS/MS. Forty-six proteins were identified based on known CHO sequences and homology to other mammalian species (e.g., mouse, human) [18].…”

Overexpression of ST6GalNAcV, a ganglioside-specific α2,6-sialyltransferase, inhibits glioma growth in vivo

“…Its involvement in the inflammatory breast cancer phenotype (15) and in breast cancer metastases to the brain (16) has been reported. Alterations in the adhesive properties of HeLa cells by modulation of ST6GalNAcV expression has also been demonstrated (17). Interestingly, there is little to no α2,6-sialytransferase (ST6Gal1) activity, the enzyme that sialylates glycoprotein-associated, N-linked oligosaccharides, in malignant glioblastomas.…”