Standardized method to minimize variability in a functional P2X(7) flow cytometric assay for a multi-center clinical trial.

BACKGROUND: Flow cytometric analysis of human P2X(7) pore activity segregates variant from common P2RX7 genotypes and may serve as a biomarker for cancer, pain, inflammation, and immune responses to infection. Standardization is needed to accommodate variable sample age and instrumentation differences in a multicenter clinical trial. METHODS: CD14-PE-stained whole blood samples were treated with YO-PRO-1 combined with a P2X(7) agonist (BzATP) or control, followed by the addition of PI after closure of the P2X(7) pore. Recalled instrument settings from previous publications were used to adapt a standardized fluorescent particle-adjusted set-up method. Experiments were performed to compare the two methods while evaluating components of systematic variability and facilitating reliable processing of samples with varied ages. RESULTS: The median YO-PRO-1 fluorescence of BzATP-treated samples had less variability when collected by the bead-adjusted method and was less influenced by the compensation strategy used. The average day-to-day coefficient of variance for assessments of P2X(7) pore activity by this method was 0.11 +/- 0.04, and the exclusion of nonviable cells was found to accommodate samples aged up to 4 days after phlebotomy. The bead-adjusted set-up method produced measurements differing by only 2.0% +/- 1.5% on two analog cytometers, and within similar decades when comparing analog to digital instruments. CONCLUSIONS: These results provide a standardized method for quantitative flow cytometric analysis of P2X(7) receptor phenotypes in blood monocytes with minimal intralaboratory variation and potential for interlaboratory comparisons that can greatly facilitate multicenter functional genomic clinical studies.

Combination study of 1,24(S)-dihydroxyvitamin D2 and chemotherapeutic agents on human breast and prostate cancer cell lines.

BACKGROUND: Vitamin D compounds are important modulators of cellular proliferation and differentiation, with potential utility as anticancer drugs. 1,24(S)-Dihydroxyvitamin D2 [1,24(OH)2D2] is a naturally occurring active vitamin D compound with low calcemic activity. MATERIALS AND METHODS: We evaluated the growth inhibitory effects of 1,24(OH)2D2 on LNCaP prostate cancer and MCF-7 breast cancer cells. 1,24(OH)2D2 was evaluated alone and in paired combination with nine chemotherapeutic agents. Drug interactions were analyzed using the median-effect/isobologram method. Combination index values were used to characterize the interactions as synergistic, additive, or antagonistic. RESULTS: In MCF-7 cells, 1,24(OH)2D2 produced synergistic effects with doxonrubicin and cisplatin and additive effects with busulfan, etoposide, tamaxifen, 5-fluorouracil and carboplatin. In LNCaP cells, 1,24(OH)2D2 produced a synergistic effect with carboplatin and additive effects with doxorubicin, busulfan, paclitaxel and etoposide. CONCLUSION: We conclude that 1,24(OH)2D2 may have therapeutic value in the treatment of prostate and breast cancers, alone or in combination with chemotherapeutic agents.

Persistent corticotropin-releasing factor(1) receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32.

Brain corticotropin-releasing factor (CRF) systems integrate various responses to stress. Pathological responses to stress may result from errors in CRF receptor regulation in response to changes in synaptic CRF levels. To establish an in vitro model to study brain CRF receptors, we characterized the CRF-induced modulation of CRF(1) receptors in the human neuroblastoma cell line, IMR-32. Treatment with CRF decreased CRF(1) receptor binding and desensitized CRF-induced increases in cAMP. The decrease in binding had an EC(50) of approximately 10 nM, was maximal by 30 min, and was blocked by the CRF receptor antagonist [D-Phe(12), Nle(21,38), C(alpha)-MeLeu(37)]CRF(12-41). The desensitization was homologous as vasoactive intestinal polypeptide-induced increases in cAMP were unchanged, and elevation of cAMP did not alter CRF(1) receptor binding. Treatment with CRF for up to 24 h did not alter CRF(1) receptor mRNA levels, suggesting that a posttranscriptional mechanism maintains the decrease in receptor binding. Interestingly, recovery of CRF receptor binding and CRF-stimulated cAMP production was only partial following exposure to 100 nM CRF. In contrast, receptor binding recovered to control levels following exposure to 10 nM CRF. These data suggest that exposure to high doses of CRF result in permanent changes characterized by only partial recovery. Identifying the mechanisms underlying this partial recovery may provide insights into mechanisms underlying the acute and chronic effects of stress on CRF receptor regulation.