Some chemical modulators of
cytochrome P4501A1,
Cyp1A1, expression also perturb the activity of
serine palmitoyltransferase, SPT, a heterodimeric
protein responsible for catalyzing the first reaction in
sphingolipid biosynthesis. The effect of altered SPT activity on
Cyp1A1 expression has generally been attributed to changes in the composition of bioactive
sphingolipids, generated downstream in the SPT metabolic pathway, but the precise mechanism remains poorly defined. A generally accepted model for chemical-induced transactivation of the
Cyp1A1 gene involves intracellular signaling mediated by
proteins including the arylhydrocarbon receptor, AhR, whose interaction with the 90 kilo Dalton
heat shock protein, Hsp90, is essential for maintaining a high affinity ligandbinding receptor conformation. Because
ligand-induced
Cyp1A1 expression is important in the bioactivation of environmentally relevant compounds to genotoxic derivatives capable of perturbing cellular processes, binding to Hsp90 represents an important regulatory point in the cytotoxicity process. In the present study, based on evidence that indicates subunit 1 of
serine palmitoyltransferase, SPT1, interacts with Hsp90, both
ligand-induced
Cyp1A1 transactivation and capacity for proliferation were evaluated using the wild type
Glioma LN18 human
brain cancer cell line and its recombinant counterparts expressing green fluorescent SPT1 fusion
proteins. Exposure to the prototypical
Cyp1A1 inducer,
3-methylcholanthrene, 3-MC, resulted in the translocation of SPT1 from a primarily cytoplasmic domain to sites of focal adhesion complexes. Immunolabel for Hsp90, which was dispersed throughout the cell, became primarily cytoplasmic, while the distribution of AhR remained unaffected. When compared to the wild type, cells transfected with recombinant SPT1-GFP vectors had significantly attenuated levels of 3-MC-induced
Cyp1A1 mRNA, as determined by quantitative reverse transcription PCR. Although all the
Glioma cell lines exhibited mitogenic proliferative response in dose response assay with the potent
Cyp1A1 inducers 3-MC, 2,3,7,8-
tetrachlorodibenzo-p-dioxin (
TCDD) and
benzo [k] fluoranthene, BKF, only the recombinant cell line designated - 75SPT1-GFP, which was transfected with a mutant deletion of SPT1, retained its proliferative capacity at the highest PAH doses used in this study. The results suggest that overexpressing SPT1 as a green fluorescent fusion
protein has a modulating effect on the transactivation of
Cyp1A1. This is possibly due to SPT1 interacting with Hsp90 to modulate AhR-Hsp90 interaction, and altering downstream events such as in downregulating the transactivation and metabolic activity of
Cyp1A1. This is supported by the fact that the -75SPT1-GFP recombinant cell line, with much lower capacity for
Cyp1A1 induction, exhibited sustained mitogenic response to high doses of AhR
ligands, but not the
Cyp1A1 inducible wild type. Conceivably, the effect mediated by SPT1 on the AhR signaling pathway is an important underlying factor contributing to variability in
Cyp1A1 gene expression and consequently, cytotoxic response to environmentally relevant compounds that pose risk to human health.