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Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog.

Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J - BMC Biotechnol. (2007)

Bottom Line: Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15.These results are consistent with previous studies on the functionalities of cdkl3 and cox15.For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Biotechnology Unit, Building 14A, Room 170, Bethesda, MD 20892, USA. pratikj@mail.nih.gov

ABSTRACT

Background: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.

Results: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.

Conclusion: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.

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Median expression ratios for cdkl3 and cox15 from four different spotted cDNA microarray slides. The median expression ratio is calculated from 3 or more gene-specific spots on a single slide. The error bars indicate the range in expression ratios observed for a given slide.
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Figure 2: Median expression ratios for cdkl3 and cox15 from four different spotted cDNA microarray slides. The median expression ratio is calculated from 3 or more gene-specific spots on a single slide. The error bars indicate the range in expression ratios observed for a given slide.

Mentions: Using clustering algorithms, the genes were organized into groups [14,15]. Principle component analysis (PCA) and gap statistic were used to estimate the number and size of groups inherently present in the data. Results of these algorithms indicated groups of 8, 9, 13, or 14 were likely to form. With this information, self-organizing maps (SOMs) and hierarchical clustering were then applied to the data to segregate the genes into distinct groups. These clusters were probed to identify subsets of genes with relevance to cellular growth based on known or proposed functionalities related to cell cycle regulation, apoptosis, and/or signal transduction. Genes that could be categorized in this manner were then interrogated further based on the level of differential expression with a sampling of the results shown in Table 1. The ratios listed in Table 1 were determined from a sample size of at least 12; based on the number of spots per slide and the number of slides analyzed. Of the genes listed, cdkl3 [GenBank: NM016508] and cox15 [GenBank: NM078470] were selected for further analysis. As shown in Figure 2, both cdkl3 and cox15 had expression levels greater than 1, indicating higher expression in the suspension HeLa cell line than in the attached HeLa cell line.


Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog.

Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J - BMC Biotechnol. (2007)

Median expression ratios for cdkl3 and cox15 from four different spotted cDNA microarray slides. The median expression ratio is calculated from 3 or more gene-specific spots on a single slide. The error bars indicate the range in expression ratios observed for a given slide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164945&req=5

Figure 2: Median expression ratios for cdkl3 and cox15 from four different spotted cDNA microarray slides. The median expression ratio is calculated from 3 or more gene-specific spots on a single slide. The error bars indicate the range in expression ratios observed for a given slide.
Mentions: Using clustering algorithms, the genes were organized into groups [14,15]. Principle component analysis (PCA) and gap statistic were used to estimate the number and size of groups inherently present in the data. Results of these algorithms indicated groups of 8, 9, 13, or 14 were likely to form. With this information, self-organizing maps (SOMs) and hierarchical clustering were then applied to the data to segregate the genes into distinct groups. These clusters were probed to identify subsets of genes with relevance to cellular growth based on known or proposed functionalities related to cell cycle regulation, apoptosis, and/or signal transduction. Genes that could be categorized in this manner were then interrogated further based on the level of differential expression with a sampling of the results shown in Table 1. The ratios listed in Table 1 were determined from a sample size of at least 12; based on the number of spots per slide and the number of slides analyzed. Of the genes listed, cdkl3 [GenBank: NM016508] and cox15 [GenBank: NM078470] were selected for further analysis. As shown in Figure 2, both cdkl3 and cox15 had expression levels greater than 1, indicating higher expression in the suspension HeLa cell line than in the attached HeLa cell line.

Bottom Line: Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15.These results are consistent with previous studies on the functionalities of cdkl3 and cox15.For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Biotechnology Unit, Building 14A, Room 170, Bethesda, MD 20892, USA. pratikj@mail.nih.gov

ABSTRACT

Background: Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.

Results: Two genes, cyclin-dependent kinase like 3 (cdkl3) and cytochrome c oxidase subunit (cox15), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of cdkl3 had a greater impact than that of cox15. Moreover, flow cytometric analysis indicated that cells with an insert of cdkl3 were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of cox15 was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than cdkl3. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either cdkl3 or cox15.

Conclusion: These results are consistent with previous studies on the functionalities of cdkl3 and cox15. For instance, the effect of cdkl3 on cell growth is consistent with its homology to the cdk3 gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to cox15 expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.

Show MeSH
Related in: MedlinePlus