Quantification and Glucocorticoid Regulation of Glucocorticoid Receptor Transcripts in Two Human Leukemic Cell Lines

Abstract

We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.

Glucocorticoid down-regulation of rat glucocorticoid receptor does not involve differential promoter regulation

“…However, if this was the case, we would expect that the proportion of GR mRNA containing exon 1 1 would be decreased by glucocorticoids, and this was not apparent even though effects of glucocorticoids upon thymus size and cellularity are significant at 72 h. Third, and most likely, all the mRNA variants may be similarly regulated in all cells. Pedersen and Vedeckis have recently shown that although the magnitude of glucocorticoid regulation of mouse GR mRNA variants (including 1A) differs between variants, the overall direction of regulation (up or down) is dependent on cell type and does not differ between mRNA variants (Pedersen et al 2003). Thus, in vivo, although we cannot rule out differential regulation of promoter activity over the short term (24 h), there is no differential regulation apparent over 72 h.…”

Glucocorticoid receptor transcriptional isoforms and resistance in multiple myeloma cells

“…The remaining primers for regular PCR and sequencing were designed with Primer3 software (33) available online. 3 Primers for real-time PCR quantification of promoters 1A1, 1A2, 1A3, 1B, and 1C were identical to previous study (34). The other primers were designed with Primer Express software (Applied Biosystems, Foster City, CA).…”

Transcriptional control of the glucocorticoid receptor: CpG islands, epigenetics and more

“…There seems to be also a poorly understood statistical link between the 5'UTR and 6 3' splice variants produced. One of the first studies to address this question showed that 7 exon 1A3, and to a lesser extend 1B and 1C contribute most to the expression of GR-α 8 isoform [24]. By comparing the most abundant exon 1 containing transcripts (1A, 1B 1C) 9…”

“…Page 10 of 32 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 are more sensitive to GC induced apoptosis, and 1A3-transcripts were shown to be the 7 most GC responsive of all first exons investigated [16,24]. Although 1A containing 8 transcripts correspond to only about 10% of all GR transcripts [42], their contribution to 9 the tissue-specific response to GC treatment was considered essential [16,24].…”

“…Although 1A containing 8 transcripts correspond to only about 10% of all GR transcripts [42], their contribution to 9 the tissue-specific response to GC treatment was considered essential [16,24]. The 10 human promoter 1A also has a functional binding site for Interferon Regulatory Factors 11 (IRF-1 and IRF-2), however IFNγ stimulation of CEM-C7 cells did not increase 1A 12 transcript levels, nor did it alter their susceptibility to GC-mediated apoptosis [45].…”