To explore this possibility, genomic DNA from PC-3 and PC-3M cells was digested with EcoRI, BamHI, and PstI, electrophoresed on an agarose gel and subjected to Southern blot analysis with MxA cDNA (supplemental Fig. S1). PC-3 and PC-3M showed identical patterns of hybridization, which indicated www.selleckchem.com/products/ABT-888.html that the difference in expression of MxA in PC-3 cells and PC-3M cells was not the result of a major genomic deletion or rearrangement. Induction of MxA Expression by Interferon��To determine whether IFN-�� can induce MxA expression in PC-3 and PC-3M cells, cells were treated with recombinant IFN-�� and subjected to immunocytochemical analysis using anti-MxA antibody and DAPI nuclear counterstaining (Fig. 2, A and B). Consistent with the Western blot result, this assay detected MxA protein only in untreated PC-3 and not in untreated PC-3M cells (compare upper left panels in Fig.

2, A and B). After exposure to IFN-��, the level of MxA protein increased substantially in PC-3, whereas MxA protein became detectable for the first time in PC-3M cells (compare lower left panels in Fig. 2, A and B). Western blotting (not shown) confirmed the IFN-induced increase in MxA protein expression in both cell lines. This evidence demonstrated that PC-3M cells were able to respond to IFN, consistent with the Southern blot result (supplemental Fig. S1) indicating that the MxA gene was intact in PC-3M cells. This experiment also showed that the IFN signaling pathway was still active in PC-3M cells, ruling out the possibility that lack of MxA expression in PC-3M cells was due to an inability to respond to IFN stimulation.

FIGURE 2. IFN induction of MxA expression and inhibition of motility in PC-3 and PC-3M cells. A and B, fluorescence immunocytochemistry of MxA. PC-3 (panels A) and PC-3M (panels B) were grown for 24 h on coverslips in the presence or absence of 1000 international … Effect of MxA on Motility of PC-3M Cells��The constitutive expression of MxA in PC-3 cells and absence of expression in PC-3M cells suggested the possibility that MxA might suppress some aspect of metastatic behavior. Furthermore, it had been reported that type I IFN can reduce cell motility (7), one component of metastasis. To test whether PC-3 and PC-3M differed in motility, we subjected both lines to an assay that measured the ability of cells to migrate through pores in a PET membrane. Fig. 2C shows that untreated (control) PC-3M cells were considerably more motile than PC-3, Cilengitide and IFN-�� reduced PC-3M motility to a level comparable to that of untreated PC-3. Consistent with the result seen in Fig. 2A, PC-3 cells were also responsive to IFN, which reduced their motility still further.