Jesse D. Martinez

Different bile acids exhibit distinct biological effects: the tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation.

Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.

The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent.

ABSTRACT Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.

Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues.

14‐3‐3 Proteins play important roles in a wide range of vital regulatory processes, including signal transduction, apoptosis, cell cycle progression and DNA replication. In mammalian cells, 7 14‐3‐3 isoforms (β, γ, ϵ, η, σ, θ and ζ) have been identified and each of these seems to have distinct tissue localizations and isoform‐specific functions. Previous studies have shown that 14‐3‐3 protein levels are higher in human lung cancers as compared to normal tissues. It is unclear, however, which of the 14‐3‐3 isoform(s) are overexpressed in these cancers. In our study, the levels of all seven 14‐3‐3 isoforms were examined by RT‐PCR and Western blotting. We show that the message for only two isoforms, 14‐3‐3ϵ and ζ, could be detected in normal tissues. In lung cancer biopsies, however, four isoforms, 14‐3‐3β, γ, σ, and θ, in addition to 14‐3‐3ϵ and ζ, were present in abundance. The expression frequency of 14‐3‐3β, γ, σ and θ isoforms was 11, 10, 13 and 8 of the 14 biopsies examined, respectively. The data from immunohistochemical staining and Western blotting were consistent with the RT‐PCR results. Given the prevalence of elevated 14‐3‐3 expression in human lung cancers we propose that these proteins may be involved in lung cancer tumorigenesis and that specific 14‐3‐3 proteins may be useful as markers for lung cancer diagnosis and targets for therapy.

Bile acid hydrophobicity is correlated with induction of apoptosis and/or growth arrest in HCT116 cells

Faecal bile acids have long been associated with colon cancer; highly hydrophobic bile acids, which induce apoptosis, have been implicated in the promotion of colon tumours. The moderately hydrophobic chemopreventive agent ursodeoxycholic acid (UDCA) does not induce apoptosis; rather, it causes colon-derived tumour cells to arrest their growth. To investigate the relationship between bile acid hydrophobicity and biological activity we examined 26 bile acids for their capacity to induce apoptosis or alter cell growth. We found that the rapidity with which, and the degree to which, bile acids could induce apoptosis or growth arrest was correlated with their relative hydrophobicities. Of the bile acids tested, only deoxycholic acid (DCA) and chenodeoxycholic acid, the most hydrophobic bile acids tested, could induce apoptosis in less than 12 h in the human colon cancer cell line HCT116. The moderately hydrophobic bile acids hyoDCA, lagoDCA, norDCA, homoUDCA and isoUDCA induced growth arrest at 12 h but longer incubations resulted in apoptosis. Conjugation of glycine or taurine to the bile acids decreased relative hydrophobicity and eliminated biological activity in our assays. In addition, we tested a subset of these bile acids for their ability to translocate across cell membranes. When (14)C-labelled and (3)H-labelled DCA, UDCA and lagoDCA were added to cell cultures, we found only minimal uptake by colon cells, whereas hepatocytes had considerably higher absorption. These experiments suggest that hydrophobicity is an important determinant of the biological activity exhibited by bile acids but that under our conditions these activities are not correlated with cellular uptake.

A shallow parser based on closed-class words to capture relations in biomedical text

Natural language processing for biomedical text currently focuses mostly on entity and relation extraction. These entities and relations are usually pre-specified entities, e.g., proteins, and pre-specified relations, e.g., inhibit relations. A shallow parser that captures the relations between noun phrases automatically from free text has been developed and evaluated. It uses heuristics and a noun phraser to capture entities of interest in the text. Cascaded finite state automata structure the relations between individual entities. The automata are based on closed-class English words and model generic relations not limited to specific words. The parser also recognizes coordinating conjunctions and captures negation in text, a feature usually ignored by others. Three cancer researchers evaluated 330 relations extracted from 26 abstracts of interest to them. There were 296 relations correctly extracted from the abstracts resulting in 90% precision of the relations and an average of 11 correct relations per abstract.

Deoxycholic Acid Induces Intracellular Signaling through Membrane Perturbations

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-β-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.

Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation

Abstract Qi, W. and Martinez J. D. Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation. Radiat. Res. 160, 217–223 (2003). The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, β, ϵ, θ and ζ, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, σ and γ, were present only at very low levels. Immunoprecipitation data showed 14-3-3ζ could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3ζ in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G2/M-phase arrest. Moreover, 16% 14-3-3ζ antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3ζ is involved in G2 checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.

Ursodeoxycholic acid modulates histone acetylation and induces differentiation and senescence

Agents that can modulate colonic environment and control dysregulated signaling are being evaluated for their chemopreventive potential in colon cancer. Ursodeoxycholate (UDCA) has shown chemopreventive potential in preclinical and animal models of colon cancer, but the mechanism behind it remains unknown. Here biological effects of UDCA were examined to understand mechanism behind its chemoprevention in colon cancer. Our data suggests that UDCA can suppress growth in a wide variety of cancer cell lines and can induce low level of apoptosis in colon cancer cells. We also found that UDCA treatment induces alteration in morphology, increased cell size, upregulation of cytokeratin 8, 18 and 19 and E‐cadherin, cytokeratin remodeling and accumulation of lipid droplets, suggesting that UDCA induces differentiation in colon carcinoma cells. Our results also suggest significant differences in UDCA and sodium butyrate induced functional differentiation. We also report for the first time that UDCA can induce senescence in colon cancer cells as assessed by flattened, spread out and vacuolated morphology as well as by senescence marker β‐galactosidase staining. We also found that UDCA inhibits the telomerase activity. Surprisingly, we found that UDCA is not a histone deacytylase inhibitor but instead induces hypoacetylation of histones unlike hyperacetylation induced by sodium butyrate. Our results also suggest that, although UDCA induced senescence is p53, p21 and Rb independent, HDAC6 appears to be important in UDCA induced senescence. In summary, our data shows that UDCA modulates chromatin by inducing histone hypoacetylation and induces differentiation and senescence in colon cancer cells.

14-3-3γ Induces Oncogenic Transformation by Stimulating MAP Kinase and PI3K Signaling

The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3γ was shown by focus formation and tumor formation in SCID mice using 14-3-3γ transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3σ, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3γ. In addition, we found that 14-3-3γ interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3γ as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3γ induced transformation.

Regulation of DNA binding and transactivation in p53 by nuclear localization and phosphorylation

Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subcellular localization and phosphorylation status of the protein. To learn more about how these two processes determine whether p53 becomes activated, we studied the temperature sensitive murine p53, p53val135. At nonpermissive temperatures, p53val135 remains sequestered in the cytoplasm of cells which express it. Electrophoretic mobility shift assays demonstrated that, under these conditions, the protein lacked DNA binding activity. However, by shifting to the permissive temperature, p53val135 became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manner. This was accompanied by the induction of two p53 regulated genes, mdm2 and p21waf1, which indicated that p53val135 had become an active transcription factor. Two dimensional gel electrophoresis and tryptic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had become extensively phosphorylation at the N-terminus. Notably, phosphorylation at the N-terminus occurred only in the nucleus, whereas phosphorylation at the C-terminus could occur in both the cytoplasm and the nucleus. Based on these observations, we suggest that phosphorylation of p53s N-terminus is compartmentally restricted.