CLL B cells express constitutive levels of HIF-1α. (A) Immunohistochemistry of CLL and normal bone marrow for HIF-1α expression. Normal and CLL bone marrow sections were immunostained with a mouse monoclonal antibody to HIF-1α and counterstained with hematoxylin. Normal marrow cells show little or an undetectable level of HIF-1α while CLL bone marrow exhibit clusters of lymphocytes positive for HIF-1α expression with mostly nuclear staining. Figures shown are representative of 5 normal and 10 CLL bone marrows (magnification ×400). (B,C) CLL B cells express high levels of HIF-1α and its target genes under normoxia. Lysates prepared from CLL B cells (P1-6), normal PBMCs (N1), and purified normal B cells (N2) were analyzed for the expression of HIF-1α or VEGF by Western blot using specific antibodies. Expression of Glut1, another target gene of HIF-1α, is also shown in these samples (except CLL-P1) by Western blot analysis using a specific antibody to Glut1. Actin was used as the loading control. (D) Expression of HIF-1α and VEGF in CLL is positively correlated. Densitometric values of HIF-1α and VEGF expression in CLL B cells (B,C) were calculated and presented as relative expression based on the values obtained from the normal purified CD19⁺ B-cell lysates (panels B,C lane N2). Expression levels of HIF-1α and VEGF in normal B cells were arbitrarily chosen as one. Results demonstrate a generally positive association of HIF-1α and VEGF expression.