The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D 9k (CaBP) were measured by Western blot; 1,25-hydroxyvitamin D 3 24-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 ϭ P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.intestine; calcium absorption; calbindin D 9k; calcium transport channel; plasma membrane Ca-adenosinetriphosphatase INTESTINAL CALCIUM ABSORPTION and its regulation by vitamin D status have been studied for over 60 years (26). The active metabolite that controls this process is 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] (27). To date, two models have been proposed to explain this process: the facilitated diffusion model (2) and the vesicular model (23). In the facilitated diffusion model, summarized in Fig. 1, the basal rate of calcium uptake and calcium extrusion from the absorptive epithelial cell is proposed to be sufficient to accommodate the elevated rate of transport seen after vitamin D stimulation. In contrast, mathematical modeling shows that intracellular diffusion is the rate-limiting, as well as the most vitamin D responsive, step. Calbindin D 9k has been proposed as the protein that binds calcium and facilitates its diffusion between the apical and basolateral surfaces of the cell (8). The level of calbindin D 9k in the intestine is closely correlated with the efficiency of calcium absorption (2), and thus this protein plays a central role in the facilitated diffusion model. In the vesicular model, the cell uses lysosomes to sequester calcium and facilitate its movement to the basolateral membrane for extrusion (24). While calbindin D 28k (the form found in avian intestine) has been associated with lysosomes (25), the role of calbindin in this model is less clear. In the past, we have tested the facilitated diffusion model using cultures of the colonic carcinoma cell line Caco-2 (9, 11, 12, 32).Caco-2 cells are a human colon adenocarcinoma cell line that can spontaneously differentiate in culture and acquire a small intestinal phenotype, e.g., they become polarized columnar epithelial cells, they form tight junctions and domes, and they express several markers that are unique to differentiated small intestinal epithelium...

Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/ variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advancedstage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125. (Cancer Res 2006; 66(3): 1792-8)

<div>Abstract<p>Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advanced-stage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125. (Cancer Res 2006; 66(3): 1792-8)</p></div>

Supplementary Methods from Application of Bayesian Modeling of Autologous Antibody Responses against Ovarian Tumor-Associated Antigens to Cancer Detection

No abstract