While IGF-1 plays a role in early B-cell development, little is known of insulin and insulin-like growth factor-1 (IGF-1) action in post-marrow B-cells. Recently, our laboratory demonstrated that mouse and human multiple myeloma (MM) cell lines possess functional insulin receptors (IRs) and IGF-1 receptors (IGF-1Rs). In this study, we show that responsiveness to insulin and IGF-1 is more developed in human MM cell lines than in human B-lymphoblastoid cell lines. Two human MM cell lines (U266 and RPMI 8226) were compared to three B-lymphoblastoid cell lines [Epstein-Barr virus immortalized B-cells (EBV), a Burkitt lymphoma cell line (Ramos), and a non-EBV lymphoblastoid cell line (HS Sultan)]. Surface IR and IGF-1R expression, measured by flow cytometry, demonstrated that the MM cell lines expressed more IRs and IGF-1Rs than did the EBV, Ramos, or HS Sultan cell lines. In vitro receptor kinase activity of affinity-purified receptors showed that the MM cells had more phosphorylated receptors than did the EBV, Ramos, or HS Sultan cells. Intracellular receptor signaling was also markedly different between the two cell groups. Whole cell phosphorylation studies showed that MM cells possessed not only hormone-dependent receptor autophosphorylation (Mr 97,000) but also substrate phosphorylation (Mr 185,000; 60,000). The lymphoblastoid cells, while demonstrating receptor autophosphorylation (IR autophosphorylation in the EBV cell line at 200 nm hormone was similar to MM receptor phosphorylation at 2 nm), lacked hormone-responsive substrates. The MM cell lines contained significantly more hormone-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity than the B-lymphoblastoid cell lines. In the MM cells, PI 3-kinase was activated by at least 10-fold, but, in the B-lymphoblastoid cell lines, it was activated by no more than 2-fold. Hormone-responsive glucose metabolism was also greater in the MM cell lines. In the U266 cells, insulin increased lactate production 62 ± 9 and 101 ± 12% (mean ± SE) at concentrations of 2 nm and 200 nm, respectively. IGF-1 produced 72 ± 9 and 99 ± 13% increases at similar concentrations. In the 8226 cells, insulin increased lactate production 4 ± 4 and 36 ± 15% at 2 and 200 nm, respectively. IGF-1 produced a 13 ± 6 and 70 ± 18% increase. In the EBV and Ramos cells, neither hormone increased lactate production by more than 10 ± 3%. Finally, we examined the mouse MM cell line Ag8.653 and, like the human MM cell lines, it had marked IGF-1-dependent receptor and substrate phosphorylation, PI 3-kinase activation, and lactate production. In conclusion, IR and IGF-1R expression and function are markedly greater in MM cell lines compared to B-lymphoblastoid cell lines, and this suggests that further differentiation is needed to fully develop insulin- and IGF-1-signaling pathways in post-marrow B-cells.
This work was supported by NIH Grant DK-38138 (to R. A. M.) and NIH Training Grant DK 07092 (to G. G. F.).