Inhibition of pathologic immunoglobulin-free light chain production by small interfering RNA molecules

Abstract

Objective: Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal immunoglobulin light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved antiplasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference.

Materials and methods: SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the cytomegalovirus promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the American Type Culture Collection (Manassas, VA, USA). Both were treated with small interfering RNA directed specifically to the V, J, or C portions of the molecules and then analyzed by enzyme-linked immunosorbent assay, flow cytometry, and real-time polymerase chain reaction.

Results: Transfected cells were found to constitutively express detectable quantities of messenger RNA and protein Wil and, after exposure to small interfering RNAs, an ∼ 40% reduction in messenger RNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells.

Conclusions: Our results have shown that RNA interference can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma.

Figure 1

Uptake of siRNA by SP2/O-λ6 and 8226 cells. Flow cytometric analyses of (A) SP2/O-λ6 and (B) 8226 cells transfected with Alexa 488-labeled or unlabeled AllStars siRNA 24, 48, or 72 hours post treatment (regional markers were based on cells transfected with non-labeled AllStars siRNA). Numbers represent the percentage of positively transfected cells.

Figure 2

Reduction in LC mRNAs in siRNA transfected mouse and human myeloma cells. (A) SP2/O-λ6 cells treated with siRNA specific for the V or J portions of λ6 light chain Wil or to the Cλ region. (B) 8226 cells treated with siRNA directed to either the V- or C-terminus of λ LC 8226. The gene expression levels of LCs Wil and 8226 at 24, 48, and 72 hours were quantified by RT-PCR using murine GAPDH as a calibrator gene (performed in triplicate). Bars indicate the expression of treated versus sham siRNA treated cells as the mean ± SEM of 3 representative experiments.

Figure 3

Effect of siRNA on LC synthesis. (A, upper) SP2/O-λ6 and (lower) 8226 cells were cultured 24, 48, or 72 hours with siRNA oligonucleotides, as indicated. At given time points, the centrifuged supernatants were analyzed by ELISA. The data shown represent the ratio of LC concentrations within supernatants of siRNA-treated cells versus controls. For each group, ratios were averaged and analyzed by one-way ANOVA (n = 6; mean ± SEM; *, p < 0.05). (B) Cλ-specific siRNA induced reduction in plasma cell LC concentration. Flow cytometric analysis of 8226 cells transfected with siRNAs C1 λ, C2 λ (open histograms) or AllStars controls (closed histograms). Regional markers that excluded sham treated cells were set for green fluorescence. For each panel, the geometric mean of the gated cell population and the percent of cells falling within the marker region are indicated.

Figure 4

Effect of siRNA on cell viability. After exposure to V-, J-, and C- region specific siRNAs or AllStars control siRNA, transfected SP2/O-λ6 (A) and 8226 cells (B) were assessed for viability using the CellTiter Blue assay. Data were pooled from 5 and 6 experimental replicates, respectively, and analyzed by one-way ANOVA (mean ± SEM; *, p < 0.05).