Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.

The in vitro experiment was carried out following 32P-postlabeling analysis to determine the DNA-reactive bile acids present in the human body. The unconjugated and conjugated forms of cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA) and lithocholic acid (LCA) were added to calf thymus DNA followed by 1 h of incubation at 37 degrees C. After the incubation the mixture was analyzed by the nuclease P1 modification of 32P-postlabeling. Among the 12 bile acids tested, our results showed that unconjugated CDCA and LCA and the glycine and taurine conjugates of LCA (g-LCA, t-LCA) were able to bind covalently with naked DNA in vitro without intervention of any catalyst. It was also shown that bile acid alone did not give any spot on TLC. These binding reactions depended on the bile acid concentration in a linear manner. The data on the extent of binding at a concentration of 0.1 mg/ml showed values of 28.5 (t-LCA), 23.7 (g-LCA), 3.47 (LCA) and 1.32 (CDCA) adducts per 10(8) nucleotides. These reactive bile acids were also incubated with COLO 205 human colon carcinoma cells and Hep G2 human hepatocellular carcinoma cells for 24 h, but no specific DNA adduct was formed. Further, when LCA or CDCA was administered into male Fischer 344 rats by gavage at a dose of 10 mg/rat every 8 h for 3 days, no specific DNA adduct was detected in their liver or colon. Covalent DNA adducts are believed to cause alteration of the primary structure of genes, which is potentially linked to carcinogenesis. Though our preliminary data failed to detect any bile acid-related DNA adducts in the cultured cells or in rats, the results may provide a basis for assuming some of these bile acids to be potential initiators of colon cancer.

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DNA of normal mucosa and the adjacent muscular layer from 18 adults suffering from colorectal neoplasms was examined by 32P-post-labeling analysis in order to estimate the exposure of the human colon and rectum to environmental carcinogens. Colorectal DNA samples obtained from six newborns were also examined as a normal control because they were presumed to have been minimally exposed to environmental carcinogens. One common mucosa-specific DNA adduct was found in the normal colorectal wall in all adults at the level of 0.10-34.13 adducts/10(8) nucleotides (mean +/- SD: 3.64 +/- 7.92 adducts/10(8) nucleotides), however, these were absent from the newborns' colons. Although several common spots were present in the mucosa, muscular layer and newborn tissues, there was no muscular layer-specific DNA adduct. The relationship between the levels of the mucosa-specific DNA adduct in the non-cancerous part and the histological degree of malignancy was not significant. The presence of this mucosa-specific DNA adduct in adult colon suggests that the human colon is commonly exposed mainly to one environmental carcinogen. This carcinogen is supposed to originate from foods, because the incidence of colorectal carcinoma is closely linked to dietary habits and the mucosa-specific DNA adduct was not present in newborns who had never ingested food. The incidence of adult colonic cancer originating from its mucosa is high, while cases of muscular origin or in newborn colon are rare. Therefore, the mucosa-specific DNA adduct is presumably responsible for the development of colonic cancer of epithelial origin.