The Effects of Hydroquinone and Benzoquinone on the Transcription Factors PU.1, AML-1, C/EBP, C-MYB AND GATA-2 IN HL-60 CELLS
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Benzene, a common chemical solvent and component of cigarette smoke, is a ubiquitous environmental pollutant that most Canadians are routinely exposed to by inhalation. Benzene is classified as a group 1A carcinogen by the International Agency for Research on Cancer (IARC) and exposure to benzene is linked to both myeloid leukemia and aplastic anemia in humans. Benzene is metabolized to toxic reactive metabolites by CYP2E1 enzymes in the body and many studies have concluded that benzoquinone and hydroquinone are the most toxic of benzene metabolites. There are several transcription factors that play a significant role in hematopoietic differentiation. Of these transcription factors, PU.1, AML-1, C/EBP-a, c-Myb, and GATA-2 are considered the most crucial to differentiation. PU.1, AML-1, C/EBP-a, c-Myb, and GATA-2 are specific to cells in the hematopoietic system and play a critical role in hematopoietic differentiation as knockout mice deficient in any of these transcription factors possess impaired hematopoietic cells, eventually resulting in death of the animal. To test the hypothesis, that exposure of HL-60 cells to hydroquinone and benzoquinone will result in altered DNA binding activity and relative protein expression of PU.1, AML-1, C/EBP-a, c-Myb and GATA-2, HL-60 cells were plated at 5.0 x105 cells/ml in 10 ml plates and exposed to either phosphate buffered saline, or equal parts hydroquinone or benzoquinone totaling 5, 10, 15, or 25 µM parts for 20 hours. Following exposure, cells were harvested and nuclear fractions were isolated and then used in a filter plate assay to determine DNA binding of the transcription factor. Western blotting was also performed on nuclear extract samples in order to confirm protein presence. Filter plate assays revealed no significant differences in DNA binding amongst the different treatment groups when assaying for PU.1, AML-1 and C/EBP-a iiDNA binding. However, c-Myb and GATA-2 DNA binding assays revealed a decrease in DNA binding when the 25 µM benzoquinone and hydroquinone treatment group was compared to the vehicle control. Western blotting performed on the nuclear extracts demonstrated the presence of PU.1, AML-1, C/EBP-a, c-Myb and GATA-2 with no observable difference in relative protein expression. This data suggests that altered DNA binding of c-Myb and GATA-2 may play a role in benzene-mediated toxicity.
URI for this recordhttp://hdl.handle.net/1974/15914
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