In essence, the project has deviated little from the plan outlined in the original proposal. We still hope to elucidate mechanisms whereby lymphocytes kill some, but not all, tumor (or undifferentiated) cells in the absence of detectable humoral antibody. During the coming year, we shall primarily emphasize studies designed to elucidate """"""""recognition"""""""" step of the NK-target cell interaction. Since this must be a property of the target cell membrane, the plasma membranes of NKsensitive, NK-insensitive, and PMA-treated target cells will be isolated and purified by established methods. This will involve homogenization and sucrose gradient centrifugation, after which the extracted membrane proteins will be separated on SDS-PAGE. The membrane preparations will be monitored for purity by enzyme analyses and EM. If any differences in patterns become apparent, the bands in question will be eluted and characterized by standard biochemical techniques. Another approach not described in our original proposal is the possible detection of antigenic differences between NK-sensitive and NK-insensitive cells by antisera raised to these cells. Since, as a rule, antigenic determinants are lost during cell differentiation, we intend to use fetal fibroblasts, which we have already shown during the past year to be sensitive to NK lysis, to raise antisera in rabbits. If our hypothesis is correct, absorption of the antiserum with adult fibroblasts should retain antibody activity to fetal fibroblasts. The antiserum will then be used to isolate the determinants which may be involved in NK cell recognition. Theoretically, thia should relate to the distinguishing of a band we hope to delineate on SDS-PAGE. The remainder of the studies during the coming year will deal with contact inhibitory factor (CIF). CIF is found in medium conditioned by a contact-inhibited melanocytic cell line. We have observed that NK-sensitive targets cultured in the presence of CIF for four to five days regain contact-inhibited growth and become NK resistant. In addition, we have learned that PMA treatment of NK-resistant targets, renders them NK sensitive. Freeze-fracture analysis of PMA-treated targets has shown a 50% reduction of intramembranous particles associated with the external leaflet of the plasma membrane. These observations will be pursued during the coming year. (AG)
| Lavie, G; Zucker-Franklin, D (1989) Cell surface-associated proteinases in NK cell-mediated cytotoxicity: enhancement of enzyme expression is unique to activation with interferon-alpha. Cell Immunol 124:202-11 |
| Zucker-Franklin, D; Nabi, Z F (1986) Loss of intercalated membrane particles by treatment with phorbols. Proc Natl Acad Sci U S A 83:6829-33 |
| Zucker-Franklin, D; Nabi, Z F (1985) A substrate analog inhibitor for arylsulfatase reduces NK cell cytotoxicity. Biochem Biophys Res Commun 126:540-3 |
