Jennifer S. Hackbarth

Accuracy of calculated free testosterone differs between equations and depends on gender and SHBG concentration.

Serum free testosterone (fT) concentrations are often calculated, however different equations often yield discrepant results. This study explores the sources of this variability. We compared three established and two new equations that differed only by their testosterone association constants with isotope dilution equilibrium dialysis in two patient groups with different gender distributions. Equation components were examined to determine how they impacted correlation with isotope dilution equilibrium dialysis. Association constants derived for each patient group correlated best with isotope dilution equilibrium dialysis for that group and not the other set. Samples with the poorest correlation between isotope dilution equilibrium dialysis and calculated fT results had significantly higher SHBG concentrations. Regardless of equation, ≥ 25% of samples showed unacceptable deviation from isotope dilution equilibrium dialysis. Association constants and gender makeup and SHBG concentration of the patient groups used to establish an equation all significantly impact correlation with isotope dilution equilibrium dialysis. Application of many fT equations to wider populations will therefore frequently yield results that differ substantially from isotope dilution equilibrium dialysis.

Cytotoxicity of farnesyltransferase inhibitors in lymphoid cells mediated by MAPK pathway inhibition and Bim up-regulation

The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.

The Putative PAX8/PPARγ Fusion Oncoprotein Exhibits Partial Tumor Suppressor Activity through Up-Regulation of Micro-RNA-122 and Dominant-Negative PPARγ Activity.

In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P < 0.05) in all FTC versus normal, 9.2-fold in FTC versus FA (P < 0.05), and 16.8-fold (P < 0.001) in FTC + PPFP versus FTC - PPFP. Constitutive expression of PPFP in the FTC-derived cell line WRO (WRO-PPFP) caused a 5-fold increase of miR-122 expression (P < 0.05) and a striking 5.1-fold reduction (P < 0.0001) in tumor progression compared to WRO-vector cells in a mouse xenograft model. Constitutive expression of either miR-122 or a dominant-negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P < 0.001] and 1.7-fold [P < 0.03], respectively). PPFP-induced up-regulation of miR-122 expression was independent of its known dominant-negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

Performance of CEA and CA19-9 in identifying pleural effusions caused by specific malignancies

OBJECTIVES Tumor markers analysis has been proposed as a less invasive alternative for categorizing malignant and non-malignant pleural effusions. This study establishes diagnostic cutoffs for CEA and CA19-9 in pleural fluid to differentiate etiologies of effusions. DESIGN AND METHODS Pleural effusions obtained from 198 patients (100 malignant, 98 non-malignant) were analyzed for CEA and CA19-9. ROC curve analysis was performed to determine analyte cutoffs, and cutoff performance was examined in various subsets of malignancies. RESULTS CEA and CA19-9 concentrations were significantly higher in malignant effusions compared to non-malignant effusions, particularly in effusions caused by lung cancer and other cancers associated with elevated CEA and/or CA19-9 serum concentrations. Concentrations were not elevated in effusions caused by cancers not associated with serum tumor marker elevations. CONCLUSIONS Measurement of CEA and CA19-9 in pleural fluid complements cytology and other classifying tests. Performance is specifically enhanced in effusions caused by malignancies known to secrete CEA or CA19-9, and their use should be tailored to patients suspected of having those malignancies. Routine analysis of these markers is therefore not recommended in all pleural effusions. Moreover, negative results should be correlated with serum levels to assist in the clinical interpretation.

On the role of topoisomerase I in mediating the cytotoxicity of 9-aminoacridine-based anticancer agents

The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In previous work, compounds of this type were shown to catalytically inhibit topoisomerase II, leading to a G1-S phase arrest of the cell cycle and apoptosis in pancreatic cancer cells in vitro and in vivo. The present study expands the potential utility of these compounds in the development of cancer therapeutics by showing that these compounds inhibit proliferation of cell lines derived from the nine most common human cancers. Further results show that at least one of the compounds effectively stabilizes topoisomerase I-DNA adduct formation in intact cells. RNA interference experiments, however, indicate that this interaction does not contribute to the drug-induced killing of cancer cells indicating the compounds may be non-lethal poisons of topoisomerase I.

Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. The locations of most topo I phosphorylation sites have not been identified, preventing a more detailed examination of this modification. To address this issue, mass spectrometry was used to identify four topo I residues that are phosphorylated in intact cells: Ser10, Ser21, Ser112, and Ser394. Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser10 can be phosphorylated by casein kinase II, Ser21 can be phosphorylated by protein kinase Cα, and Ser112 and Ser394 can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser21 phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells.