ABSTRACT. Fanconi anemia (FA) is a recessively inherited disease characterized by bone marrow failure, congenital anomalies, chromosomal instability and hypersensitivity to crosslinking agents. Some of the cellular defects of FA are known to be responsive to the ambient oxygen concentration. We examined the responsiveness of the FA complementation group C (FAC) gene to changes in oxygen concentration using two types of human cell lines, hypoxia-responsive Hep3B hepatoma cells and Epstein-Barr virus-immortalized lymphoblasts (normal and FA complementation groups B and C). Although the expression of erythropoietin in Hep3B cells was induced in response to the hypoxia-mimicking agent CoCl2, there was no concomitant induction in FAC expression as assessed by mRNA levels and immunoprecipitable protein, and no detectable change in the cytoplasmic location of the FAC polypeptide as determined by indirect immunofluorescence. In human lymphoblasts we examined the effect of oxygen (0.1%-95% O2) on cell proliferation and FAC expression. FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen. Our results indicate that ambient oxygen is not a regulator of the FAC gene.

Keywords: Fanconi anemia, gene expression, oxygen, hypoxia, hyperoxia

Reprint requests to: Hagop Youssoufian, M.D., Department of Medicine, Hematology-Oncology Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115 USA, phone (617) 732-5464, fax (617) 739-3324, e-mail: hagop@calvin.bwh.harvard.edu.

ABSTRACT. Stimulated human peripheral blood mononuclear cells (MNC) have been shown to express both G-CSF and GM-CSF. Furthermore, G-CSF is expressed by monocytes but not lymphocytes, whereas GM-CSF is expressed largely by T lymphocytes and at low levels in monocytes/ macrophages. Here we present the effect of TPA (12-O-tetradecanoyl phorbol-13-acetate) on G-CSF and GM-CSF expression in stimulated human MNCs and T lymphocytes. We observed that TPA (30nM) decreased G-CSF mRNA levels in MNCs, while ionomycin increased G-CSF in a dose-dependent manner. TPA and ionomycin individually increased GM-CSF mRNA levels in T-lymphocytes and MNCs. Further, GM-CSF was induced synergistically by TPA plus ionomycin, whereas this combination markedly decreased G-CSF mRNA levels in MNCs. These data suggest at least two signaling pathways by which G-CSF and GM-CSF mRNA levels are modulated in a mixed population of monocytes and T lymphocytes, namely protein kinase C (PKC) and calcium. These signals seem to act synergistically in lymphocytes to increase GM-CSF, and not G-CSF mRNA levels specifically. It would also appear these signals act on MNCs in an opposing manner to decrease G-CSF mRNA levels, indicating that activation of PKC and the calcium signaling pathway lead to a cell-type specific modulation of individual cytokines and precise regulation of granulocyte production.

Keywords: granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, peripheral blood mononuclear cells, phorbol esters; mRNA expression.

Reprint requests to: William P. Hammond, M.D., Department of Medical Education, Providence Medical Center, 500 17th Ave, C-34008, Seattle, WA 98124 USA, phone (206) 320-2552, fax (206) 320-3589, e-mail: whammond@u.washington.edu.