Solar ultraviolet B (UVB) acts as both an initiator and promoter in models of multistage skin carcinogenesis. We found that, whereas UVB induces apoptosis in human papillomavirus-16 E6/7-immortalized keratinocytes, it inhibits markers of differentiation in human foreskin keratinocytes (HFK). Potential mechanisms for this differential response were examined by DNA microarray, which revealed that UVB alters the expression of three of the four human inhibitor of differentiation/DNA binding (Id) proteins that comprise a class of helix-loop-helix family of transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. These results were verified by RT-PCR and immunoblot analysis of control and UVB-irradiated primary and immortalized keratinocytes. Whereas Id1 was downregulated in both cell types, Id2 expression was upregulated in primary HFK, but not immortalized cells. In contrast, Id3 expression was significantly increased only in immortalized cells. The differential expression pattern of Id2 in response to UVB was recapitulated in reporter constructs containing the 5 0 regulatory regions of this gene. Id2 promoter activity increased in response to UVB in HFK, but not in immortalized cells. To identify the regulatory elements in the Id2 promoter that mediate transcriptional activation by UVB in HFK, promoter deletion/mutation analysis was performed. Deletion analysis revealed that transactivation involves a 166 bp region immediately upstream to the Id2 transcriptional start site and is independent of c-Myc. The consensus E twenty-six (ETS) binding site at -120 appears to mediate UVB transcriptional activation of Id2 because point mutations at this site completely abrogated this response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays verified that the Id2 promoter interacts with known Id2 promoter (ETS) binding factors Erg1/2 and Fli1, but not with c-Myc; and this interaction is enhanced after UVB exposure. Similar to the effects of UVB exposure, ectopic expression of Id2 protein in primary HFK resulted in inhibition of differentiation, as shown by decreased levels of the terminal differentiation marker keratin K1 and inhibition of involucrin crosslinking. Reduction of Id2 expression by small interfering RNAs attenuated the UVB-induced inhibition of differentiation in these cells. These results suggest that UVB-induced inhibition of differentiation of primary HFK is at least, in part, due to the upregulation of Id2, and that upregulation of Id2 by UVB might predispose keratinocytes to carcinogenesis by preventing their normal differentiation program.

Lung epithelial cells are critical in the regulation of airway inflammation in response to environmental pollutants. Altered activation of NF-kappaB is associated with expression of several proinflammatory factors in respiratory epithelial cells in response to an insult. Here we show that a low threshold dose (8 microg/ml) of the jet fuel JP-8 induces in a rat alveolar epithelial cell line (RLE-6TN) a prolonged activation of NF-kappaB as well as the increased expression of the proinflammatory cytokines TNF-alpha and IL-8, which are regulated by NF-kappaB. The up-regulation of IL-6 mRNA in cells exposed to JP-8 appears to be a reaction of RLE-6TN cells to reduce the enhancement of proinflammatory mediators in response to the fuel. Moreover, lung tissues from rats exposed to occupational levels of JP-8 by nasal aerosol also showed dysregulated expression of TNF-alpha, IL-8, and IL-6, confirming the in vitro data. The poly(ADP-ribosyl)ation of PARP-1, a coactivator of NF-kappaB, was coincident with the prolonged activation of NF-kappaB during JP-8 treatment. These results evidenced that a persistent exposure of the airway epithelium to aromatic hydrocarbons may have deleterious effects on pulmonary function.

3038 Multiple myeloma (MM), one of the most incurable hematological malignancies in adults, is a disorder of plasma cells characterized by accumulation of clonal proliferation of malignant plasma cells in the bone marrow (BM). Overexpression of beta-catenin, the downstream effector of the canonical Wnt signaling pathway, has been reported in both MM cell lines and patient samples. Activated Wnt signaling pathway has also been reported to play a critical role in progression of MM cell proliferation, thus highlighting the need for new therapeutic approaches, particularly those targeting Wnt molecular pathway. Here we report the discovery of a novel inhibitor of Wnt signaling CWP232291, which promotes degradation of beta-catenin. CWP232291 exhibits potent growth inhibitory activity in several MM cell lines (RPMI-8226, OPM-2, NCI-H929, JJN3, and EJM) with IC50 values of 13 – 73 nM. The inhibitory activity of CWP232291 on Wnt signaling is demonstrated by reporter gene assay and by its effect in down-regulation of Wnt target genes. Using HEK293 cells, CWP232291 treatment dose dependently reduces promoter activity of TOPflash induced by Wnt-3a-Conditioned Media, at a calculated IC50 value of 273 nM. Furthermore, MM cells treated with CWP232291 show downregulated expression of Wnt target genes such as survivin by triggering degradation of beta-catenin. Treatment of these cells with CWP232291 results in activation of caspase-3 and PARP cleavage, indicating induction of apoptosis. To investigate the potential in vivo anti-tumor efficacy of CWP232291, we utilized two MM tumor bearing mice models. Daily or intermittent intravenous (i.v.) administration of CWP232291 led to significant tumor growth inhibition (TGI) in OPM-2 (50 mg/kg, qdx5: regression and 100 mg/kg, biw: 95% TGI) and RPMI-8226 (100 mg/kg, qdx5: regression and 100 mg/kg tiw: 80% TGI) xenograft model with no obvious change in body weight. The anti-tumor efficacies of CWP232291 were dose-dependent and had strong correlations with degradation of beta-catenin in the tumor samples. Potent induction of apoptosis through cleavage of Caspase-3 and PARP and significant decrease of plasma M protein levels in OPM-2 tumor bearing mice were detected as early as 2 and up to 24 hours after single i.v. administration of CWP232291. Taken together, these data clearly demonstrate the impressive anti-tumor profile of CWP232291 with a good therapeutic window and suggest a potential therapeutic application of CWP232291 for the treatment of MM. Disclosures: Cha: Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Briaud:Theriac Pharmaceutical Corp.: Employment. Tenzin:Theriac Pharmaceutical Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pyon:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Chung:Choongwae Pharma Corp.: Employment. Lee:Choongwae Pharma Corp.: Employment. Oh:Choongwae Pharma Corp.: Employment. Jung:Choongwae Pharma Corp.: Employment. Pai:Choongwae Pharma Corp.: Employment. Emami:Theriac Pharmaceutical Corp.: Employment.

Wnt signaling pathway controls various cellular and developmental processes. Deregulation of this pathway, in a wide range of human solid tumors, has been well established. However, less is known about its role in etiology of hematological cancers. Some characteristics of acute myelogenous leukemia (AML) cells such as inappropriate proliferation in the absence of normal growth signals and indefinite self-renewal similar to a stem cell are thought to be due to mutations in β-catenin, the hallmark of canonical Wnt pathway, and/or activation of Wnt pathway by fusion transcription factors. For therapeutic purposes, we have screened a peptidomimetic, β-turn library of small molecules for an inhibitor of Wnt pathway. Through massive chemical modifications the lead compound CWP231904 was selected. CWP231904 showed good inhibitory activity in the Reporter Gene Assay (RGA) on Topflash promoter in SW480 and HEK293 cells. Wnt target genes, survivin and c-myc were both down regulated upon CWP231904 treatments at the level of RNA and protein in MV4-11 and HL-60 AML cells. CWP231904 treatments of both cell lines induce cleavage of caspase-3, -8, and -9 by apoptotic signals. Propidium Iodide staining of these cells show dramatic increase in Apoptotic Index. CWP231904 treatment of MV4- 11 and HL-60 cells led to degradation of β-catenin in both cell lines. Proteasome inhibitor, MG132, prevented the CWP231904 induced degradation of β-catenin in MV4-11 cells. In order to unravel the mode of action of CWP231904, we utilized Wnt3a conditioned media (CM) or an overexpressed, Estradiol (ER) inducible Dishevelled plasmid (ER-DVL-2) to activate Wnt signaling in HEK293 cell line. Immunoblotting of HEK293 cells induced with ER and treated with CWP231904 showed a nuclear migration of the phosphorylated form of DVL-2 in treated cells. Thus, our data suggest that CWP231904 targets β-catenin degradation by relocating DVL-2 from cytoplasm where it makes puncta and inactivates GSK-3β containing destruction box complex into the nucleus. Moreover, CWP231904 treatment of HEK293 cells growing in Wnt3a CM lowered the inhibitory phosphorylation of GSK3-β on Serine9 which is induced upon Wnt activation, thereby keeping GSK3-β in its active form to phosphorylate β-catenin and signal it for degradation. CWP231904 significantly reduced tumor growth in MV4-11, HL-60 and MOLM-13 xenograft models, significantly increased the life span of the SCID mice, and decreased the number of CD45+ AML cells in the bone marrow after engraftment of MV4-11 cells via intravenous injection withoutdetectable toxicity. Together, these results suggest that CWP231904 has therapeutic potential for the treatment of AML disease by inhibiting Wnt signaling pathway through destabilization of β-catenin.