Dip1 inhibits growth and gene transcription in MCF-7 breast cancer cells.

In previous studies we identified a novel gene Dipl, also designated CCNDBP1, which encodes a 42kDa helix-loop-helix (HLH) nuclear protein. Although this protein was originally identified by its ability to bind to cyclin D1 its precise biochemical functions are not known. In the present study we carried out mechanistic studies on Dip1 focusing on the human breast cancer cell line MCF-7. We found that overexpression of Dip1 in MCF-7 cells inhibited colony formation and cell proliferation. Reporter assays in MCF-7 cells indicated that Dip1 strongly inhibited the transcriptional activities of the cyclin D1, c-fos, NF-kappaB, SRE and p21cP1 promoters. Furthermore studies with truncated and mutant forms of the cyclin D1 promoter suggest that Dip1 does not act on specific transcriptional elements. Assays with mutant and truncated forms of Dip1 indicated that both the LXXLL motif and the HLH domain play important, but not exclusive, roles in these inhibitory effects. Dip1 co-immunoprecipitated with the histone deacetylase (HDAC) proteins HDAC1 and HDAC3. Nevertheless Dip1 markedly inhibited the stimulation of cyclin D1 promoter activity obtained with trichostatin A [1], an inhibitor of HDAC. Taken together these findings suggest that Dip1 functions as a general repressor of transcription. Although the precise mechanism by which Dip1 inhibits gene transcription and the growth of MCF-7 cells remain to be determined, the present results suggest that Dip1 is a candidate tumor suppressor gene.

Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells.

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.

Desmoglein genes are up-regulated in the pk mutant mouse.

Plucked (pk) is an autosomal recessive mouse mutation with a hair phenotype that arose spontaneously in the DBA/2J strain. Histological studies indicate that adult pk mutant mice lose truncal hair because of the scarring of follicles due to an apparent obstruction of the outward movement of the hair shaft within the follicular canal. We mapped the pk mutant phenotype to a 1.1cM region of chromosome 18 (between 6.6 and 7.7 cM from the centromere) using 370 backcross progeny. Within this region, among others, are genes for desmosome cadherins. Desmosome cadherins are interesting candidates because of their critical roles for cell-cell adhesion in epidermal function. Northern Blot analysis of wild-type and pk mutant mice indicates that expression of both desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) is up-regulated in the skin of mutant pk mice.