FLOW CYTOMETRIC ANALYSIS OF IN-VIVO POLYAMINE DEPRIVATION IN LEWIS-LUNG-CARCINOMA (3LL) CELLS USING THE MONOCLONAL-ANTIBODY SPM8-2

It has previously been shown that the monoclonal antibody SPM8-2 recog nizes free spermine and spermidine as well as polyamines bound by an a mide bond, In the present work it is demonstrated that this antibody a lso interacts with spermidine, spermine, and to a lesser extent N-1- a nd N-8-acetyl spermidine in an ELISA test where the polyamines are bou nd by reaction with formaldehyde, 3LL Lewis lung carcinoma cells from tumor-grafted mice were labeled with fluorescein-conjugated monoclonal antibody SPM8-2 and analyzed by flow cytometry, Both viable cells and formaldehyde-fixed and subsequently permeabilized cells showed fluore scent staining, However, most polyamines present in the cells are not directly available for antibody binding, Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting tha t some polyamines are co-localized with DNA and RNA, Antibody labeling of the cells was prevented by addition of free spermine, 3LL, cells f rom tumors of mice treated by a polyamine depleting regimen had decrea sed intracellular spermidine levels and bound less antibody when compa red to untreated controls, After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor c ells from untreated mice, while their RNA content was similar. In cont rast, fluorescent staining after DNase digestion was only slightly aff ected by the treatment with a polyamine depleting regimen. This sugges ts that the pools of polyamines which are co-localized with RNA are de pleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is neces sary to reveal specific compartments of the polyamines and to demonstr ate qualitative and semi-quantitative differences of their distributio n within cells. (C) 1997 Wiley-Liss, Inc.