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Effect of nickel(II) on DNA-protein interactions
T P Coogan1, D M Latta, R J Imbra
1Institute of Environmental Medicine, New York University Medical Center, Tuxedo 10987.
Abstract:
Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using 3H-thymidine and 35S-methionine, respectively. Nuclei were exposed to NiCl2 in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37 degrees C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (3H) retained on each filter was normalized to protein (35S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl2 resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl2 inhibits specific DNA-protein interactions.
Insights
Nickel chloride (NiCl2) exposure inhibits DNA-protein interactions (DPI), potentially impacting cancer development. This study demonstrates NiCl2
Area of Science:
- Biochemistry
- Molecular Biology
- Carcinogenesis
Background:
- Alterations in DNA-protein interactions (DPI) are implicated in cancer development.
- The precise mechanism of nickel-induced carcinogenesis remains unclear, though nickel is known to affect DPI.
Purpose of the Study:
- To investigate the effect of nickel chloride (NiCl2) on DNA-protein interactions in Chinese hamster ovary (CHO) cells.
- To determine if NiCl2 inhibits specific DNA-protein binding relevant to carcinogenesis.
Main Methods:
- Utilized a nitrocellulose filter assay to measure DPI in intact CHO cells and isolated nuclei.
- Employed radiolabeling of DNA (3H-thymidine) and proteins (35S-methionine) for quantitative analysis.
- Used a band shift assay with a cloned satellite DNA sequence to assess specific protein-DNA binding inhibition by NiCl2.
Main Results:
- Exposure to increasing, non-cytotoxic concentrations of NiCl2 caused a dose-dependent decrease in overall DPI in both whole cells and isolated nuclei.
- Nickel significantly inhibited specific protein binding to a satellite DNA probe in band shift assays.
- Both assays indicated that NiCl2 inhibits specific DNA-protein interactions at physiological pH.
Conclusions:
- Nickel chloride demonstrably inhibits specific DNA-protein interactions.
- These findings suggest a potential mechanism for nickel's role in carcinogenesis through interference with critical DNA-protein binding events.