Signals from CD30 D1 drive increased expression of p21^WAF1/CIP1 and growth arrest in ALCL cells.

<p>(A) Schematic representation of all retroviral constructs showing the position of domains (D1, D2 and D3) of hCD30 downstream of m4-1BB extracellular and transmembrane (TM) domains. Numbers indicate CD30 residues relative to their position in full-length hCD30. (B) Karpas-299 cells were stably transduced with the constructs indicated and surface expression of m4-1BB detected by flow cytometry (open histograms); filled histograms represent staining with isotype control. (C) Karpas-299 transductants were stimulated for 6 or 24 hours with 4-1BBL or CD30L or left untreated for 24 hours and p21^WAF1/CIP1 detected by immunoblot; actin is shown as a loading control. Blots are representative of two independent experiments. To determine the role of CD30 domains in triggering growth arrest, Karpas-299 (D) or Michel (E) cells transduced with the plasmids indicated, were stimulated for 72 hours and [³H]-thymidine uptake determined as a measure of cell cycling. Data are normalised to maximum growth inhibition (that induced by CD30L, set at 100%) and to minimum inhibition (medium alone; 0%) to show relative % inhibition induced by CD30L, 4-1BBL or control (CD70). Data in panels D and E are pooled from 3 of 3 (D) or 2 of 2 (E) experiments and show means and SEM. One-way ANOVA was performed on 4-1BBL-stimulated data sets across all experiments and was significant for Karpas-299 (p<0.0001) and Michel (p<0.018) cells. Statistical analyses using the Students two-tailed t-test were therefore performed comparing the means of individual groups; *p<0.05, **p<0.005, n.s. p>0.05.