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Heavy metals stimulate human LINE-1 retrotransposition.

Kale SP, Moore L, Deininger PL, Roy-Engel AM - Int J Environ Res Public Health (2005)

Bottom Line: Reproducible results showed that mercury (HgS), cadmium (CdS), and nickel (NiO) increase the activity of L1 by an average of three (3) fold p<0.001.This observation is the first to link several carcinogenic agents with the increased retrotransposition activity of L1 as an alternate mechanism of generating genomic instability contributing to the process of carcinogenesis.Our results demonstrate that mobile element activation must be considered as one of the mechanisms when evaluating genomic damage/instability in response to environmental agents.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Xavier University of Louisiana, 1 Drexel Dr. New Orleans, LA 70125, USA.

ABSTRACT
L1 and Alu elements are among the most active retroposons (mobile elements) in the human genome. Several human diseases, including certain forms of breast cancer and leukemia, are associated with L1 and Alu insertions in functionally important areas of the genome. We present data demonstrating that environmental pollutants, such as heavy metals, can stimulate L1 retrotransposition in a tissue culture system using two different types of assays. The response to these agents was equivalent when using a cell line with a stably integrated L1 vector (genomic) or a by introducing the L1 vector by transient transfection (episomal) of the cell. Reproducible results showed that mercury (HgS), cadmium (CdS), and nickel (NiO) increase the activity of L1 by an average of three (3) fold p<0.001. This observation is the first to link several carcinogenic agents with the increased retrotransposition activity of L1 as an alternate mechanism of generating genomic instability contributing to the process of carcinogenesis. Our results demonstrate that mobile element activation must be considered as one of the mechanisms when evaluating genomic damage/instability in response to environmental agents.

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Effect of different compounds on L1 retrotransposition activity (transient transfection assay).A. Metals stimulate L1 retrotransposition in a transient transfection assay: NeoR colonies from separate L1 transfections (black bar) treated with different doses of HgS, CdS or NiO (X axis) are shown. An unrelated plasmid with neomycin resistance was used as a transfection and toxicity control (white bar). The no treatment (0 dose) for each experiment was used as the 100%. In a similar manner as used for the L1-stable assay described in the text, the data were adjusted for toxicity (gray bar). Three independent assays in triplicate (n=9) were performed in HeLa cells and error bars indicate standard deviations. Statistically significant differences are indicated relative to the no treatment [t-test p<0.01(*), p<0.001(**)]. All the metals tested with this assay show a stimulation of L1 retrotransposition comparable to that observed in the L1-stable assay.B. Schematic of time line of the L1 transient assay: The L1-vector is transfected into cells that were seeded the previous day (150,000 in T75). Immediately after transfection (3 hour) cells are treated with the metal for 48 hours. Treatment is removed and cells are grown under selection for 2 weeks before staining. Total duration of the assay is 2.5 weeks.
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f3-ijerph-02-00014: Effect of different compounds on L1 retrotransposition activity (transient transfection assay).A. Metals stimulate L1 retrotransposition in a transient transfection assay: NeoR colonies from separate L1 transfections (black bar) treated with different doses of HgS, CdS or NiO (X axis) are shown. An unrelated plasmid with neomycin resistance was used as a transfection and toxicity control (white bar). The no treatment (0 dose) for each experiment was used as the 100%. In a similar manner as used for the L1-stable assay described in the text, the data were adjusted for toxicity (gray bar). Three independent assays in triplicate (n=9) were performed in HeLa cells and error bars indicate standard deviations. Statistically significant differences are indicated relative to the no treatment [t-test p<0.01(*), p<0.001(**)]. All the metals tested with this assay show a stimulation of L1 retrotransposition comparable to that observed in the L1-stable assay.B. Schematic of time line of the L1 transient assay: The L1-vector is transfected into cells that were seeded the previous day (150,000 in T75). Immediately after transfection (3 hour) cells are treated with the metal for 48 hours. Treatment is removed and cells are grown under selection for 2 weeks before staining. Total duration of the assay is 2.5 weeks.

Mentions: HeLa cells (ATCC CCL2) were grown in a humidified, 5% CO2 incubator at 37°C in Earl’s minimal essential medium (EMEM). EMEM was supplemented with 10% fetal bovine serum. HeLa cells were seeded in T-75 flasks at a density of 1.5 × 105 cells/flask and grown for 20 hours prior to transfection. Cells were transfected with the Lipofectamine Plus (InVitrogen) for three hours using 1μg of either the L1 or 0.3μg the neomycin control plasmid with 18μl of the plus reagent and 12μl of Lipofectamine, following the manufacturer’s protocol. The transfection mix was aspirated and replaced with the complete media supplemented with the appropriate dose of the compound evaluated. After a 48 hour period, the treatment was removed and the cells were grown for two weeks using selection media (containing 400μg/ml G418) to obtain the neomycin resistant (neoR) colonies. Cell colonies were fixed and stained for 30 minutes with crystal violet (0.2% crystal violet in 5% acetic acid and 2.5% isopropanol). A schematic of the time line is shown on Figure 3B.


Heavy metals stimulate human LINE-1 retrotransposition.

Kale SP, Moore L, Deininger PL, Roy-Engel AM - Int J Environ Res Public Health (2005)

Effect of different compounds on L1 retrotransposition activity (transient transfection assay).A. Metals stimulate L1 retrotransposition in a transient transfection assay: NeoR colonies from separate L1 transfections (black bar) treated with different doses of HgS, CdS or NiO (X axis) are shown. An unrelated plasmid with neomycin resistance was used as a transfection and toxicity control (white bar). The no treatment (0 dose) for each experiment was used as the 100%. In a similar manner as used for the L1-stable assay described in the text, the data were adjusted for toxicity (gray bar). Three independent assays in triplicate (n=9) were performed in HeLa cells and error bars indicate standard deviations. Statistically significant differences are indicated relative to the no treatment [t-test p<0.01(*), p<0.001(**)]. All the metals tested with this assay show a stimulation of L1 retrotransposition comparable to that observed in the L1-stable assay.B. Schematic of time line of the L1 transient assay: The L1-vector is transfected into cells that were seeded the previous day (150,000 in T75). Immediately after transfection (3 hour) cells are treated with the metal for 48 hours. Treatment is removed and cells are grown under selection for 2 weeks before staining. Total duration of the assay is 2.5 weeks.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814692&req=5

f3-ijerph-02-00014: Effect of different compounds on L1 retrotransposition activity (transient transfection assay).A. Metals stimulate L1 retrotransposition in a transient transfection assay: NeoR colonies from separate L1 transfections (black bar) treated with different doses of HgS, CdS or NiO (X axis) are shown. An unrelated plasmid with neomycin resistance was used as a transfection and toxicity control (white bar). The no treatment (0 dose) for each experiment was used as the 100%. In a similar manner as used for the L1-stable assay described in the text, the data were adjusted for toxicity (gray bar). Three independent assays in triplicate (n=9) were performed in HeLa cells and error bars indicate standard deviations. Statistically significant differences are indicated relative to the no treatment [t-test p<0.01(*), p<0.001(**)]. All the metals tested with this assay show a stimulation of L1 retrotransposition comparable to that observed in the L1-stable assay.B. Schematic of time line of the L1 transient assay: The L1-vector is transfected into cells that were seeded the previous day (150,000 in T75). Immediately after transfection (3 hour) cells are treated with the metal for 48 hours. Treatment is removed and cells are grown under selection for 2 weeks before staining. Total duration of the assay is 2.5 weeks.
Mentions: HeLa cells (ATCC CCL2) were grown in a humidified, 5% CO2 incubator at 37°C in Earl’s minimal essential medium (EMEM). EMEM was supplemented with 10% fetal bovine serum. HeLa cells were seeded in T-75 flasks at a density of 1.5 × 105 cells/flask and grown for 20 hours prior to transfection. Cells were transfected with the Lipofectamine Plus (InVitrogen) for three hours using 1μg of either the L1 or 0.3μg the neomycin control plasmid with 18μl of the plus reagent and 12μl of Lipofectamine, following the manufacturer’s protocol. The transfection mix was aspirated and replaced with the complete media supplemented with the appropriate dose of the compound evaluated. After a 48 hour period, the treatment was removed and the cells were grown for two weeks using selection media (containing 400μg/ml G418) to obtain the neomycin resistant (neoR) colonies. Cell colonies were fixed and stained for 30 minutes with crystal violet (0.2% crystal violet in 5% acetic acid and 2.5% isopropanol). A schematic of the time line is shown on Figure 3B.

Bottom Line: Reproducible results showed that mercury (HgS), cadmium (CdS), and nickel (NiO) increase the activity of L1 by an average of three (3) fold p<0.001.This observation is the first to link several carcinogenic agents with the increased retrotransposition activity of L1 as an alternate mechanism of generating genomic instability contributing to the process of carcinogenesis.Our results demonstrate that mobile element activation must be considered as one of the mechanisms when evaluating genomic damage/instability in response to environmental agents.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Xavier University of Louisiana, 1 Drexel Dr. New Orleans, LA 70125, USA.

ABSTRACT
L1 and Alu elements are among the most active retroposons (mobile elements) in the human genome. Several human diseases, including certain forms of breast cancer and leukemia, are associated with L1 and Alu insertions in functionally important areas of the genome. We present data demonstrating that environmental pollutants, such as heavy metals, can stimulate L1 retrotransposition in a tissue culture system using two different types of assays. The response to these agents was equivalent when using a cell line with a stably integrated L1 vector (genomic) or a by introducing the L1 vector by transient transfection (episomal) of the cell. Reproducible results showed that mercury (HgS), cadmium (CdS), and nickel (NiO) increase the activity of L1 by an average of three (3) fold p<0.001. This observation is the first to link several carcinogenic agents with the increased retrotransposition activity of L1 as an alternate mechanism of generating genomic instability contributing to the process of carcinogenesis. Our results demonstrate that mobile element activation must be considered as one of the mechanisms when evaluating genomic damage/instability in response to environmental agents.

Show MeSH
Related in: MedlinePlus