Figure 4. Overexpression of STAT3 upregulates caspase3 and induces apoptosis in MM-1 and CLL cells.

(A) MM-1 cells were transfected via electroporation with a mammalian vector that coexpressed the full-length STAT3 sequence and GFP or with the same vector expressing only GFP (empty vector) and stimulated with IL-6. Using double staining for annexinV/PI, we detected the cellular apoptosis rate via flow cytometry 24 hours after the transfection. As shown, at a transfection rate of 42%, 85% of CLL cells transfected with STAT3 underwent apoptosis compared to a 59% apoptosis rate of cells transfected with the empty vector. A representative Figure from 3 different experiments is depicted. In all experiments apoptosis rate was higher in MM-1 cell transfected with recombinant STAT3 DNA (P< 0.001; paired t-test). (B) Relative expression of STAT3 and caspase3 mRNA assessed via qRT-PCR in MM-1 cells transfected with STAT3 and stimulated with IL-6. Compared to control untransfected cells, STAT3 and caspase3 RNA levels were markedly upregulated. RNA levels of STAT3 and caspase3 in STAT3-transfected MM-1 cells. As shown, transfection of MM-1 cells with STAT3 induced a 7.5-fold increase in STAT3 and a 2.5-fold increase in caspase3 RNA levels. The means ± SD of gene expression fold change from 3 different experiments are depicted. (C) Upper panel: Relative expression of STAT3 and caspase3 mRNA assessed using qRT-PCR in CLL cells infected with a lentivirus harboring the human STAT3 gene. CLL cells were infected with the full-length STAT3 gene sequence. As shown, overexpression of STAT3 (≥ 30% infection efficiency) induced a 13.5-fold increase in STAT3 and a 6-fold increase in caspase3 RNA levels. The means ± SD of gene expression fold change from 3 different experiments using the same patient’s cells are depicted. Lower panel: Protein levels of STAT3 and uncleaved and cleaved Caspase3 of the same patient’s cells assessed by western immunoblotting and quantitated by densitometry. As shown, STAT3 levels increased by 0.6-fold and cleaved Caspase3 levels by 0.8-fold compared with their corresponding levels in cells infected with the empty vector. Similar results were obtained using CLL cells from two other patients with low lymphocyte counts. (D) Apoptosis rate of the CLL cells infected with the full-length STAT3 gene or the empty vector assessed by flow cytometry 24 hours after infection. As shown, at an infection rate of 30%, the apoptosis rate of CLL cells transfected with STAT3 was 70.8% whereas that of cells infected with the empty vector was 59.7%. Similar results were obtained using cells from two other patients with low lymphocyte counts.