Arsenic trioxide (ATO) is a first-line anti-cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long-term exposure. At 24 h after exposure, ATO (0.01-1 µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non-tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell-cycle-associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low-dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose- and time-dependent manner. Low-dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO-increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO-induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced cell proliferation and cell cycle modulation from G1 to S phase.

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Recent advances in analytical cytometry have improved diagnostic tools for the study of erythropoiesis in anemic patients and resolution of the differential diagnosis in diseases of the erythron. This unit presents three applications of red blood cell (RBC) analysisquantitation of fetal red cells (see Basic Protocol 1), F cell enumeration (see Basic Protocol 2), and F reticulocyte analysis (see Basic Protocol 3)-that improve diagnostic precision, sensitivity, and specificity, and provide better laboratory indicators of therapeutic efficacy in a variety of hematologic and obstetric disorders. Such advances also include the measurement and quantitation of RBC hemoglobins and their relative nucleic acid levels. These advances not only promise to improve diagnostic accuracy and laboratory precision over techniques such as the traditional manual reticulocyte counting method and the Kleihauer-Betke stain method for evaluating fetomaternal hemorrhage (FMH), but also serve as tools for newer assays of anemia diagnosis and improved clinical outcomes. In addition to the primary methods, supporting techniques for preparing spiked controls (see Support Protocol 1), setting up a fetal hemoglobin acquisition protocol (see Support Protocol 2), and assaying reticulocytes using thiazole orange (see Support Protocol 3) are also presented. BASIC PROTOCOL 1 QUANTITATION OF FETAL RED CELLS BY FLOW CYTOMETRY Flow cytometric methods are more sensitive and precise than routine visual counting and reliably detect low levels of fetal RBCs in circulating maternal blood. The following protocol for the detection of fetal hemoglobin (HbF)-containing RBCs uses a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC), a method for the intracytoplasmic staining of red blood cells, and WinList or any other equivalent Software (Davis et al., 1998; Chen et al., 2000); however, the method can be easily adapted for use with anti-HbF monoclonal antibody reagents conjugated to other fluorochromes and with other data-analysis software. Materials Whole-blood samples anticoagulated with EDTA or other suitable anticoagulant (test within 4 hr of collection or store ≤72 hr at 4°C) Fetaltrol stabilized three-level controls (Caltag Laboratories) or spiked control blood samples (see Support Protocol 1) PBS/0.1% BSA (see recipe) 0.05% glutaraldehyde (see recipe) 0.1% Triton X-100 (see recipe) Anti-HbF antibody dilution (see recipe) 1% formaldehyde fixative (see recipe) 12 × 75-mm disposable polystyrene tubes (Falcon) with rack DAC II automatic cell washer (Baxter) or equivalent (optional) Multiparameter flow cytometer with 488-nm excitation and filters for detection of green and orange fluorescence WinList Software (Verity Software House; http://www.vsh.com) or equivalent Additional reagents and equipment for counting cells (APPENDIX 3A) and acquisition of data (see Support Protocol 2) Supplement 28