Novel Functional Association of Serine Palmitoyltransferase Subunit 1-A Peptide in Sphingolipid Metabolism with Cytochrome P4501A1 Transactivation and Proliferative Capacity of the Human Glioma LN18 Brain Tumor Cell Line

Abstract

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 ligand-binding 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 var...

Expression of C-Terminal Modified Serine Palmitoyltransferase-1 Alters Chemosensitivity of Inflammation-Associated Human Cancer Cell Lines

“…It is possible that the modulation of CBX-mediated transcriptional expression of Cyp3A4 by SPTLC1, via a mechanism-based inhibition effect, combined with systemic metabolic inhibition due to Geldanamycin, may be contributing to the increased chemosensitivity exhibited by the Glio-CSPT recombinant cells used in the current study. This observation corresponds to a previous report implicating SPTLC1 expression in mechanism-based inhibition of the transcriptional expression of Cyp1A1 in 3-MC treated cells [6].…”

“…In all samples tested, Cyp3A4 transcript level in CBX treated cells is markedly decreased in Glio-CSPT recombinant Glioma cells, compared to the other cell lines, consistent with the ability of the C-terminal modified SPTLC1 variant to alter Cyp450 gene transcript level (6). Together, the data in Figure 7 and Figure 9, as well as Table 1, show that in only the Glio-CSPT cell construct, SPTLC1 expression alters CBX mediated transcriptional ex- 50 value computed from the CBX treatment divided by that of the CBX/SAS treatment.…”

“…In contrast, a decrease of the CI 50 value from 25 μM to 12 μM for Glio-CSPT cells treated with CBX/Geldanamycin is indicative of increase in chemosensitivity to CBX. Coincidentally, the Glio-CSPT cells are reported to have attenuated transcriptional expression of Cyp1A1 when treated with 3-MC [6]. In Figure 9, similar to decreased transcriptional expression of Cyp1A1 in 3-MC treated cells, the transcriptional expression of Cyp3A4 is also attenuated in Glio-CSPT recombinant cells when treated with CBX.…”

“…The compound is a potent inducer, through AhR, of the transcriptional expression of Cyp1A1 [39]. Although SPTLC1 can affect transcriptional expression of Cyp1A1 [6], panel C of Figure 7 indicates that the presence of 3-MC did not significantly modulate chemosensitivity to CBX. Geldanamycin is known to inhibit the transcriptional expression of Cyp450 through binding to Hsp90 and hence disrupting AhR receptor-mediated transcriptional expression of Cyp1A1 [59].…”

“…Generally, interaction with the Hsp90 molecular chaperone is essential to the proper folding of the proteins and their assembly into modular functional complexes [43]. Incidentally, SPTLC1 is an Hsp90 binding partner, an interaction that has been implicated in altered transcriptional expression of Cyp1A1 and metabolism of cognate AhR ligands including 3-MC [6] [44].…”