Figure 3
Figure 3. Role of class IA isoforms in BCR-induced and constitutive PI3K signaling. (A) Jeko-1 cells were pretreated for an hour with isoform-selective inhibitors (1 µM) as indicated, followed by IgM stimulation with 10 µg/mL anti-human IgM F(ab') fragments for 10 minutes. Phospho-Akt (thr308) levels were compared by western blotting with nonstimulated and IgM-stimulated Jeko-1 controls. P-Syk (tyr525/526) was used as a marker of BCR activation. GS-1101 (p110δ-selective) blocked p-Akt production, whereas A66 (p110α-selective) and TGX-221 (p110β-selective) did not. (B) Real-time PCR and western blotting confirming increased expression of PIK3CA and p110α in Granta519 MCL cell lines compared with the lymphoblastoid cell line NcNc. (C) Western blot comparing downstream effects of GS-1101 (1 μM) and GDC-0941 (1 μM), at 2 time points demonstrating incomplete and nonsustained effect of GS-1101 on p-Akt, p-GSK3β, and p-S6. (D) Western blots were performed with serum-starved (4 hours) Granta519 cells treated with GS-1101, GDC-0941, and combinations of GS-1101 with A66 or TGX-221 (all 1 µM), for 2 hours, confirming the effect of p110α on constitutive PI3K activation. (E) Comparison of 50% inhibition/inhibitory concentration of GS-1101 and GDC-0941 for the 4 class I isoforms. GS-1101 is highly p110δ-selective, whereas p110α is predominantly p110α/δ-selective. The activity of both inhibitors against p110γ is comparable. (F) Western blot showing expression of p-Akt (t308) and the class IA catalytic unit isoforms in Jeko-1 and Granta519. (G) Greater growth inhibition is seen in Granta519 and Jeko-1 with GDC-0941 compared with GS-1101, whereas A66 has a minimal effect. (H) Dot plots showing greater cytotoxicity (ATP assay) with GDC-0941 compared with GS-1101 in 12 primary MCL samples with significant toxicity at and above 0.1 µM. (I) Western blot showing p-Akt (thr308) expression in the same 12 MCL cell suspensions. (J) Comparative cytotoxicity (ATP assay) of GDC-0941 and GS-1101 in 3 healthy B cells showing somewhat greater, but not statistically significant, cytotoxicity at all concentrations with GDC-0941. *P < .05, **P < .01.

Role of class IA isoforms in BCR-induced and constitutive PI3K signaling. (A) Jeko-1 cells were pretreated for an hour with isoform-selective inhibitors (1 µM) as indicated, followed by IgM stimulation with 10 µg/mL anti-human IgM F(ab') fragments for 10 minutes. Phospho-Akt (thr308) levels were compared by western blotting with nonstimulated and IgM-stimulated Jeko-1 controls. P-Syk (tyr525/526) was used as a marker of BCR activation. GS-1101 (p110δ-selective) blocked p-Akt production, whereas A66 (p110α-selective) and TGX-221 (p110β-selective) did not. (B) Real-time PCR and western blotting confirming increased expression of PIK3CA and p110α in Granta519 MCL cell lines compared with the lymphoblastoid cell line NcNc. (C) Western blot comparing downstream effects of GS-1101 (1 μM) and GDC-0941 (1 μM), at 2 time points demonstrating incomplete and nonsustained effect of GS-1101 on p-Akt, p-GSK3β, and p-S6. (D) Western blots were performed with serum-starved (4 hours) Granta519 cells treated with GS-1101, GDC-0941, and combinations of GS-1101 with A66 or TGX-221 (all 1 µM), for 2 hours, confirming the effect of p110α on constitutive PI3K activation. (E) Comparison of 50% inhibition/inhibitory concentration of GS-1101 and GDC-0941 for the 4 class I isoforms. GS-1101 is highly p110δ-selective, whereas p110α is predominantly p110α/δ-selective. The activity of both inhibitors against p110γ is comparable. (F) Western blot showing expression of p-Akt (t308) and the class IA catalytic unit isoforms in Jeko-1 and Granta519. (G) Greater growth inhibition is seen in Granta519 and Jeko-1 with GDC-0941 compared with GS-1101, whereas A66 has a minimal effect. (H) Dot plots showing greater cytotoxicity (ATP assay) with GDC-0941 compared with GS-1101 in 12 primary MCL samples with significant toxicity at and above 0.1 µM. (I) Western blot showing p-Akt (thr308) expression in the same 12 MCL cell suspensions. (J) Comparative cytotoxicity (ATP assay) of GDC-0941 and GS-1101 in 3 healthy B cells showing somewhat greater, but not statistically significant, cytotoxicity at all concentrations with GDC-0941. *P < .05, **P < .01.

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