Subcellular localization and growth effects of the CD44ΔEx1 variant overexpressed in the CD44⁻ GCB-DLBCL cell line BJAB. (A) No CD44 mRNA was detected by RT-PCR performed using primers CD44-F (exon 2) and CD44-R (exon 18) in total RNA extracted from the microdissected CD10⁺ GC B cells from reactive tonsils (2 preparations), whereas all the IGHSμ/CD44 translocation-positive cases showed the presence of CD44ΔEx1 mRNA; a representative translocation-positive case is shown (GL47). A vertical white line has been inserted in the bottom panel to indicate a repositioned gel lane. (B) 5′-RACE analysis detected the Iμ-CD44ΔEx1 hybrid transcripts in all 9 IGHSμ/CD44 translocation-positive cases; the 5′-RACE sequence (299 bp) from a representative translocation-positive case is shown (GL47). Stop codons (TGA) are underlined. The Iμ-CD44ΔEx1 ORF is not predicated to result in a fusion protein, as the CD44 exon 2 has an in-frame stop codon upstream of the predicted start codon ATG at nucleotide number 254 (boxed by solid line). The Iμ-CD44ΔEx1 ORF is predicted to encode for the CD44ΔEx1 protein starting from the ATG at nucleotide 254 (boxed by solid line) with a Kozak sequence²⁹ ; the ATG codon at nucleotide 183 (boxed by dotted line) has a downstream in-frame stop codon (underlined by dotted line). (C) Nucleotide sequences of the CD44ΔEx1 ORF (219 bp) and predicted amino acid sequences of the protein (73 amino acids) are shown. Exon boundaries are marked with an arrow. Compared with CD44s (standard form; wild-type), the resultant CD44ΔEx1 protein lacks the first 62 amino acids at the N-terminus, including the 20 amino acids of the leader peptide. The CD44ΔEx1 protein also lacks the C-terminus of CD44s resulting from an altered reading frame starting from the junction of exons 2 and < 17b, which eliminates the transmembrane and intracellular domains. (D) When cloned in-frame with GFP at the C-terminus of the pmaxFP-Green-N vector and overexpressed in BJAB cells, CD44ΔEx1-GFP tagged protein was localized in the cytoplasm and nucleus instead of the plasma membrane, where CD44s-GFP was expressed. Representative images of cells taken by confocal laser microscopy are shown. The images were captured by the Zeiss LSM 510 (Carl Zeiss Microimaging GmbH) laser scanning microscope system (60× oil objective). The images in superimposed and split-image mode were displayed and exported as imaging files by LSM Image Browser software (Carl Zeiss Microimaging GmbH). Merged images were generated from the original confocal images using Adobe Photoshop software. (E-F) Overexpression of CD44ΔEx1 in stably transfected BJAB cells significantly enhanced the cell proliferation rate as recorded over 5 days of incubation (mean values of 3 separate experiments ± SD are shown; E) and clonogenic ability in methylcellulose as monitored after 6 days of incubation (F). The images in panel F were captured by the Nikon Eclipse TS100 inverted microscope (Nikon) and the Nikon DSU1 0507 11331 digital camera system and the images were acquired by ACT-2U software. The magnifications are written at the right side of each panel. Results similar to those in panels D through F were obtained when CD44ΔEx1 cloned in-frame with GFP at the N-terminus of the pmaxFP-Green-C vector was used for the aforementioned functional studies (results not shown).