A combination of two drugs afforded remarkable protection from intestinal neoplasia in APC(Min/+) mice, a murine model of human familial adenomatous polyposis (FAP). One of the drugs was sulindac, a prototypical non-steroidal anti-inflammatory drug with established chemopreventative activity. The second drug was EKI-569, a newly developed, irreversible inhibitor of the epidermal growth factor receptor kinase. Although 100% of the untreated APC(Min/+) mice developed approximately 20 polyps, nearly half the mice treated with these two agents developed no polyps at all. These results suggest a powerful strategy for the chemoprevention of human colonic neoplasia.
Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination. As a test case, a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted. Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype. Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras. The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.
To identify genes that mediate transforming growth factor- (TGF-) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF- in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-.T he transforming growth factor- (TGF-) signal transduction pathway is involved in numerous biological processes (1-7). These processes include those regulating cell birth, cell death, differentiation, invasion, angiogenesis, and immunity. Therefore, disruption of the TGF- pathway has predictably been reported to occur in numerous tumor types. Genetic alterations of components of this pathway are particularly common in cancers of the colon and pancreas (8)(9)(10)(11)(12)(13)(14).TGF- ligands bind to receptor kinases on the cell surface, leading to phosphorylation of the receptor-phosphorylated Smad proteins (R-Smads). Once phosphorylated, these Smads interact with Smad 4 and translocate to the nucleus where the Smad complex binds to specific DNA sequences in conjunction with other nuclear proteins that regulate gene expression (For reviews,. Some of the genes that are thereby activated by TGF- family members have been identified in Xenopus and invertebrate systems (4,(18)(19)(20). However, knowledge of the genes that are regulated by TGF- in mammalian cells is just beginning to emerge (21-24). In the current work, we have established a useful system for studying the effects of TGF- in colorectal cancer cells and used this system, in conjunction with more conventional ones, to identify and study such genes. Materials and Methods Generation of Inducible Lines.A tetracycline (tet)-off system was used to establish inducible lines as described (25). The inducible TRII expression vector was constructed by cloning a restriction fragment containing an influenza hemagglutinin-tagged TRII ORF into pBI-MCS-EGFP (25). To construct the inducible DEC1͞GFP expression vector, the PCR-amplified human DEC1 ORF was inserted into the EcoR1 site of pEGFP-N1 (CLON-TECH). The fused DEC1͞GFP ORF was then subcloned into pTRE2 (CLONTECH). The expression constructs were cotransfected with pTK-Hyg (CLONTECH) into DLD-tet cells that constitutively express tTA (25). Single clones were isolated after selection with Genetic...
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