Metastatic and non-metastatic colorectal cancer (CRC) cells induce host metalloproteinase production in vivo.

Numerous studies have demonstrated the persistent localization of matrix metalloproteinase (MMP) expression to the interface between invading human colorectal cancer (CRC) cells and surrounding stroma supporting a role for MMPs in CRC invasion and metastasis. The present study sought to determine whether CRC cells of varying metastatic potential would have differential effects on host MMP release. Subcutaneous CRC tumors were generated in BALB/c nude mice using three CRC cell lines: SW480, SW620, and the highly metastatic SW620S5 clone. Representative samples from the subcutaneous CRC were then orthotopically implanted on the cecum of recipient nude mice. Subcutaneous and cecal tumors were analyzed for MMP expression via zymography, western blot, and RT-PCR. In vitro, none of the three cell lines expressed MMP-2 nor MMP-9. In contradistinction, the subcutaneous tumors expressed limited amounts of MMP-2 and MMP-9 while the cecal tumors expressed significant amounts of MMP-2 and MMP-9 as well as other smaller members of the MMP family. MMP-9 mRNA and protein was confirmed as host in origin by RT-PCR with mouse specific primers and a mouse MMP-9 molecular weight of 105 kDa as determined by zymography and western blot analysis. In situ hybridization also localized the mRNA for MMP-9 to the host stromal cells. In conclusion, CRC cells appear incapable of producing MMP-2 and MMP-9 in vitro but are capable of up-regulating host MMP production in vivo. Enhanced host MMP-9 production in metastatic CRC cell-derived subcutaneous and cecal tumors suggests that metastatic colon cells may acquire the expression of important MMP regulating factor(s) in vivo.

Hairpin coding end opening is mediated by RAG1 and RAG2 proteins.

Despite the importance of hairpin opening in antigen receptor gene assembly, the molecular machinery that mediates this reaction has not been defined. Here, we show that RAG1 plus RAG2 can open DNA hairpins. Hairpin opening by RAGs is not sequence specific, but in Mg2+, hairpin opening occurs only in the context of a regulated cleavage complex. The chemical mechanism of hairpin opening by RAGs resembles RSS cleavage and 3' end processing by HIV integrase and Mu transposase in that these reactions can proceed through alcoholysis. Mutations in either RAG1 or RAG2 that interfere with RSS cleavage also interfere with hairpin opening, suggesting that RAGs have a single active site that catalyzes several distinct DNA cleavage reactions.