Preliminary study of immunomagnetic quantification of circulating tumor cells in patients with advanced disease.

OBJECTIVES: To enumerate the amount of circulating tumor cells (CTCs) in patients with advanced prostate cancer and to investigate the relationship between these numbers, prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) expression, and clinical parameters. METHODS: Whole blood was collected in proprietary CellSave tubes. Mononuclear cell fractions were isolated using epithelial cell antibody-coated magnetic nanoparticles. On one half of each immunomagnetically enriched cell fraction, automated fluorescent microscopy was used to identify the epithelial tumor cells. From the remainder of each sample, RNA extraction, cDNA synthesis, and polymerase chain reaction amplification of PSA and PSM were performed. RESULTS: Eighty-four patients with advanced prostate cancer submitted 130 samples for analysis. Intact CTCs were identified in 62% of samples; 83.3% of CTC-positive and 0% of CTC-negative samples were reverse transcriptase-polymerase chain reaction positive for PSA and PSM (P = 0.001). A significant positive correlation was found between the CTC number and PSA (r = 0.49), alkaline phosphatase (r = 0.47), and lactate dehydrogenase (r = 0.55) levels, and a significant negative correlation with hemoglobin (r = -0.35). The initial Gleason grade, prior therapy, current therapy, and type of metastasis (bone, soft tissue) did not correlate significantly with the CTC number. CONCLUSIONS: The presence of intact CTCs and the expression of PSA and PSM demonstrated robust agreement. The tumor cell numbers reflected current disease status and correlated significantly with the clinical disease indicators of PSA, hemoglobin, and liver function tests. These findings warrant further investigation of the diagnostic and prognostic value of enumerating intact CTCs.

Inability of serotonin to activate the c-Jun N-terminal kinase and p38 kinase pathways in rat aortic vascular smooth muscle cells.

BACKGROUND: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells. RESULTS: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10(-9) - 10(-5) M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10(-5) M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin. CONCLUSION: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells.