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Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress.
Invest Ophthalmol Vis Sci. 2002 Oct; 43(10):3251-64.IO

Abstract

PURPOSE

The response of lens systems to oxidative stress is confusing. Antioxidative defense systems are not mobilized as expected, and unanticipated defenses appear important. Therefore, mouse lens cell lines conditioned to survive different peroxide stresses have been analyzed to determine their global changes in gene expression.

METHODS

The immortal mouse lens epithelial cell line alphaTN4-1 was conditioned to survive 125 microM H2O2 (H cells) or a combination of both 100 microM tertiary butyl hydroperoxide (TBHP) and 125 microM H2O2 (HT cells), by a methodology previously described. The total RNA was isolated from the different cell lines and analyzed with oligonucleotide mouse expression microarrays. Four microarrays were used for each cell line. Microarray results were confirmed by real-time RT-PCR.

RESULTS

A new cell line resistant to both 125 microM H2O2 and 100 micro M TBHP was developed, because cells resistant to H2O2 were killed by TBHP. Analysis of classic antioxidative enzyme activities showed little change between cells that survive H2O2 (H) and those that survive H2O2 and TBHP (HT). Therefore, the global change in gene expression in these cell lines was determined with gene expression microarrays. The fluorescent signal changes of the genes within the three cell lines, H, HT, and control (C), were analyzed by statistical methods including Tukey analysis. It was found that from the 12,422 gene fragments and expressed sequence tags (ESTs) analyzed--based on a one-way ANOVA with a stringent cutoff of one false positive per 1000 genes and correcting for microarray background and noise--approximately 950 (7.6%) genes had a significant change in expression in comparing the C, H, and HT groups. A small group of antioxidative defense genes were found in this population, including catalase, members of the glutathione (GSH)-S-transferase family, NAD(P)H menadione oxidoreductase 1, and the ferritin light chain. The remaining genes are involved in a broad spectrum of other biological systems. In the HT versus H comparison, only a few genes were found that had increased expression in the HT line compared with expression in the H line, including GSH-S-transferase alpha 3 and hephaestin. Many genes that are frequently considered antioxidative defense genes, including most of the GSH peroxidases, unexpectedly showed little change.

CONCLUSIONS

An unusual and generally unexpected small group of antioxidative defense genes appear to have increased expression in response to H2O2 stress. Cell lines resistant to H2O2 do not appear to survive challenge with another type of peroxide, TBHP, a lipid peroxide prototype. However, acquisition of TBHP resistance by H cells was found to be accompanied by significantly amplified expression of only a few additional antioxidative defense genes. Many of the amplified genes do not appear to be involved with antioxidative systems, reflecting the complexity of the cells' response to oxidative stress.

Authors+Show Affiliations

Department of Ophthalmology, Columbia University, New York, New York 10032, USA. as42@columbia.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

12356832

Citation

Spector, Abraham, et al. "Differential Amplification of Gene Expression in Lens Cell Lines Conditioned to Survive Peroxide Stress." Investigative Ophthalmology & Visual Science, vol. 43, no. 10, 2002, pp. 3251-64.
Spector A, Li D, Ma W, et al. Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress. Invest Ophthalmol Vis Sci. 2002;43(10):3251-64.
Spector, A., Li, D., Ma, W., Sun, F., & Pavlidis, P. (2002). Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress. Investigative Ophthalmology & Visual Science, 43(10), 3251-64.
Spector A, et al. Differential Amplification of Gene Expression in Lens Cell Lines Conditioned to Survive Peroxide Stress. Invest Ophthalmol Vis Sci. 2002;43(10):3251-64. PubMed PMID: 12356832.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress. AU - Spector,Abraham, AU - Li,Dayu, AU - Ma,Wanchao, AU - Sun,Fang, AU - Pavlidis,Paul, PY - 2002/10/3/pubmed PY - 2002/11/26/medline PY - 2002/10/3/entrez SP - 3251 EP - 64 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 43 IS - 10 N2 - PURPOSE: The response of lens systems to oxidative stress is confusing. Antioxidative defense systems are not mobilized as expected, and unanticipated defenses appear important. Therefore, mouse lens cell lines conditioned to survive different peroxide stresses have been analyzed to determine their global changes in gene expression. METHODS: The immortal mouse lens epithelial cell line alphaTN4-1 was conditioned to survive 125 microM H2O2 (H cells) or a combination of both 100 microM tertiary butyl hydroperoxide (TBHP) and 125 microM H2O2 (HT cells), by a methodology previously described. The total RNA was isolated from the different cell lines and analyzed with oligonucleotide mouse expression microarrays. Four microarrays were used for each cell line. Microarray results were confirmed by real-time RT-PCR. RESULTS: A new cell line resistant to both 125 microM H2O2 and 100 micro M TBHP was developed, because cells resistant to H2O2 were killed by TBHP. Analysis of classic antioxidative enzyme activities showed little change between cells that survive H2O2 (H) and those that survive H2O2 and TBHP (HT). Therefore, the global change in gene expression in these cell lines was determined with gene expression microarrays. The fluorescent signal changes of the genes within the three cell lines, H, HT, and control (C), were analyzed by statistical methods including Tukey analysis. It was found that from the 12,422 gene fragments and expressed sequence tags (ESTs) analyzed--based on a one-way ANOVA with a stringent cutoff of one false positive per 1000 genes and correcting for microarray background and noise--approximately 950 (7.6%) genes had a significant change in expression in comparing the C, H, and HT groups. A small group of antioxidative defense genes were found in this population, including catalase, members of the glutathione (GSH)-S-transferase family, NAD(P)H menadione oxidoreductase 1, and the ferritin light chain. The remaining genes are involved in a broad spectrum of other biological systems. In the HT versus H comparison, only a few genes were found that had increased expression in the HT line compared with expression in the H line, including GSH-S-transferase alpha 3 and hephaestin. Many genes that are frequently considered antioxidative defense genes, including most of the GSH peroxidases, unexpectedly showed little change. CONCLUSIONS: An unusual and generally unexpected small group of antioxidative defense genes appear to have increased expression in response to H2O2 stress. Cell lines resistant to H2O2 do not appear to survive challenge with another type of peroxide, TBHP, a lipid peroxide prototype. However, acquisition of TBHP resistance by H cells was found to be accompanied by significantly amplified expression of only a few additional antioxidative defense genes. Many of the amplified genes do not appear to be involved with antioxidative systems, reflecting the complexity of the cells' response to oxidative stress. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/12356832/Differential_amplification_of_gene_expression_in_lens_cell_lines_conditioned_to_survive_peroxide_stress_ L2 - https://iovs.arvojournals.org/article.aspx?volume=43&issue=10&page=3251 DB - PRIME DP - Unbound Medicine ER -