A procedure is described to isolate nonhistone proteins of malignant cell nuclei for the purpose of testing certain of its biologic activities. Walker rat carcinoma 256 cells were disrupted by N2 cavitation, the nuclei isolated by differential centrifugation, the chromatin was purified, and the DNA separated from chromatin protein by chromatography on Bio-Gel A 5m in the presence of 4 M guanidine-HCl. Further separation of histone and nonhistone proteins was achieved with CM-Sephadex C-50, eluting alkylated nonhistones with 0.4 M guanidine-HCl in 0.01 M phosphate buffer and subsequent elution of histones in 2 M guanidine-HCl in 0.1 M acetic acid. Nonhistone and histone proteins were obtained in good yield and satisfactory purity. The histone and nonhistone proteins were characterized by amino acid analysis, macromolecular composition, and gel electrophoresis. A fraction containing tumor nonhistone proteins was mitogenic to endothelial cells, and, when implanted into the rabbit cornea, caused proliferation of vascular endothelium and new blood vessel formation. Tumor angiogenesis factor was thus demonstrated to be associated with a fraction also containing nonhistone proteins of malignant cell nuclei. Histone fractions had no activity in this bioassay.
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