Analysis of the involvement of mesothelial cells in the antineoplastic activity of mIFN-α₁. (A) Phase-contrast and immunofluorescence images of representative polygonal EGFP-positive mesothelial cells obtained from peritoneal fragments of EGFP-LV–treated SCID mice. EGFP-positive cells were visualized using a Carl Zeiss inverted fluorescence microscope (Axiovert 200; Carl Zeiss, Jena, Germany) with a 40×/0.55 NA objective, photographed using an Olympus camera (Camedia C3020; Olympus, Center Valley, PA), visualized and adjusted using the CorelDRAW Graphics Suite X3 (Corel Corporation, Ottawa, ON). Original magnifications ×40. (B) TRAIL up-regulation in mIFN-α₁–treated murine mesothelial cells. RT-PCR analyses of murine IRF-7 (mIRF-7), TRAIL (mTRAIL), and β-actin (mβ-act, control) transcript expression were performed using RNA extracted from primary mesothelial cells 48 hours after native mIFN-α₁ (250 IU/mL) or mock treatment. Up-regulation of mIRF-7 and mTRAIL was observed in mIFN-α₁–treated but not in mock-exposed cells, suggesting that mesothelial cells may contribute to IFN-modulated antineoplastic activity. Lanes: 1, RNA extracted from untreated; 2, mock-treated; 3, mIFN-α₁–treated mesothelial cells; 4, positive control, RNA from MBL-2 cells exposed to 500 IU/mL recombinant mIFN-α₁. (C) In vivo TRAIL expression. RT-PCR analyses were performed using RNA extracted from peritoneal fragments of control and mIFN-α₁-LV–treated PEL/SCID mice. Up-regulation of mTRAIL was observed in mIFN-α₁-LV–treated but not in control mice, suggesting that host microenvironment may play a role in IFN-stimulated antineoplastic activity. Expression of murine β-actin is shown as control. Lanes: 1, RNA extracted from peritoneal fragments of medium-injected; 2, EGFP-LV–treated; 3 to 7, mIFN-α₁–treated PEL/SCID mice; 8, positive control, RNA from MBL-2 cells exposed to 500 IU/mL recombinant mIFN-α₁. (D) Role of TRAIL in IFN-induced apoptosis of CRO-AP/3 cells. Density plot histograms of flow cytometric analysis of a representative experiment show the percentage of early (annexin V–positive and PI-negative bottom circle) and late (annexin V– and PI-positive top circle) apoptosis in CRO-AP/3 cells 12 hours after mock or mIFN-α₁ (250 IU/mL) treatments, or 12 hours after coculture with mock- or mIFN-α₁ (250 IU/mL)–treated mesothelial cells in the presence of a control or a blocking anti-mTRAIL mAb. Treatment of CRO-AP/3 cells with mIFN-α₁ did not substantially affect the level of apoptosis, whereas coculture with mIFN-α₁–treated mesothelial cells significantly increased programmed cell death in CRO-AP/3 cells. CRO-AP/3 apoptosis was efficiently inhibited by the blocking anti-mTRAIL mAb, whereas an isotype-matched control mAb did not influence apoptosis induction, indicating that mesothelial cells activated by IFN-α induced apoptosis in CRO-AP/3 cells in a TRAIL-dependent manner.