David A. Stanley, John H. Frenster, and Demetrios A. Rigas
Department of Medicine, Stanford University School of Medicine, Palo Alto, California, and Division of Experimental Medicine, University of Oregon Medical School, Portland, Oregon.
E-mail: frenster@euchromatin.net
When added to otherwise quiescent human lymphocytes in vitro, phytohemagglutinin (PHA) causes a rapid de-repression of RNA synthesis, with subsequent protein and DNA synthesis and cell division. Neither the site of action nor the mechanism of action of PHA is known. Highly purified PHA in the form of the glycoprotein octamer was exposed for 2 wk to tritium gas. The resultant highly tritiated PHA (1.4 mc/mg) was incubated at a concentration of 0.01 mg/ml with resuspended cells from the buffy-coat layers of normal human blood, and these were sampled at periods of time ranging from 15 min to 24 hr . Incubated cells were sedimented and fixed without washing, and uniform ultrathin sections were prepared for electron microscope radioautography. Quantitative methods relating observed grain counts to the areas of observed subcellular organelles were employed. Localization of grains within lymphocytes was increased over 12-fold, and within granulocytes and monocytes was increased over 40-fold, as compared with immediately adjacent extracellular areas. Within lymphocytes, grains were present over both cytoplasm and nucleus, and additional grains were localized to the plasma membrane and the nuclear membrane. Within the nucleus of lymphocytes, grains were localized almost exclusively over condensed masses of heterochromatin. Extended microfibrils of active euchromatin did not display significant numbers of grains. These data suggest that PHA localizes within repressed heterochromatin during gene de-repression in human lymphocytes. Purified PHA is a polyanion (1964. Ann. N.Y. Acad. Sci, 113:800), and this molecular property may determine the preference of PHA for localization within electrostatically neutral heterochromatin, rather than within euchromatin, which already contains an excess of polyanions (1965. Nature, 206: 680).
Supported in part by grants CA-10174 and AM-01006 from the NIH and by contract AT(45-1)-581 from the AEC.
1. Stanley DA, Frenster JH, and Rigas DA, "Localization of 3H-Phytohemagglutinin within Human Lymphocytes and Monocytes", "Proceedings of the Fourth Annual Leukocyte Culture Conference, 1969", (McIntyre OR, ed.), pp. 1-11, (1971), New York: Appleton-Century-Crofts.
2. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Annals of the New York Academy of Science, vol. 277: pp. 45-51 (1976).