cAMP regulates alternative splicing of CD44 in ATRA-resistant NB4-LR1 cells.

<p>(<b>A</b>) <i>Schematic representation of the genomic structure of CD44 gene.</i> Constitutively (grey) or alternatively (dark grey) spliced exons, and position of exon-specific forward and reverse (arrows) primers used in RT-PCR are shown. (<b>B</b>) <i>Splicing pattern of CD44 in NB4 cells</i>. <i>CD44</i> sequence was amplified by RT-PCR using exon specific forward primers (v2 to v10) in combination with a constant exon 16 specific reverse primer. PCR products were analyzed on agarose gel stained by ethidium bromide, cloned and sequenced. NB4 cells express variant exons v3 and v6 as a single exon, and exon v9 in combination with v10 (<i>CD44</i>v9-10). The upper band in the “v9” lane corresponds to a new <i>CD44</i> variant with a 127 bp intronic sequence inclusion (<i>CD44</i>v9-In13-10). The expression of single exon v10 is shown in the <i>“</i>v10” lane. The asterisk corresponds to a non specific band (“v3” lane). (<b>C</b>, <b>D</b>) <i>Splicing pattern of CD44 in resistant NB4-LR1 cells.</i> Cells were mocked (−) or treated (+) with 8-CPT-cAMP (150 µM). <i>(C) CD44</i> sequence was first amplified by RT-PCR using primers spanning the alternatively splice insertion site (forward primer in constant exon 3 and reverse primer in constant exon 16). Both <i>CD44</i> standard (<i>CD44</i>s) and spliced isoforms (<i>CD44</i>v) were induced by 8-CPT-cAMP. (D) <i>CD44</i> sequence was further amplified using variant exon specific forward primers (v2 to v10) and a reverse primer in the constant exon 16. Only the expression of <i>CD44</i>v9-10 and <i>CD44</i>v9-In13-10 variants is induced by 8-CPT-cAMP. The expression of exon v10 is also shown (<i>“</i>v10” lane).