Supernatants from cultures of phytohemagglutinin (PHA)-stimulated human lymphocytes inhibit the division of certain cells in tissue culture. This effect of lymphocyte culture supernatant was routinely estimated by the rate of tritiated thymidine incorporation into DNA. In this system dilutions of 1:20 of the proliferation inhibitory factor (PIF) reduced DNA synthesis to 10% to 20% of that in control cultures. At dilutions of less than 1:640, PIF generally inhibited DNA synthesis by greater than 50%. Consistent with the decrease in DNA synthesis, cell cultures growing in the presence of PIF had fewer mitotic figures and lower total cell density than did control cultures. Supernatants from cultures established with dead leucocytes and PHA, extracts of fresh leucocytes, or PHA alone failed to produce this effect. A lesser degree of inhibitory activity also was present in supernatants from “unstimulated” lymphocyte cultures. Inhibition of proliferation in susceptible cultures was not accompanied by loss of viability; and cells from treated cultures excluded trypan blue, retained the ability to re-attach to glass, were capable of division and formed microcolonies when cloned. In addition to HeLa cells, susceptible cell cultures included human amnion, HEp 2, and KB cells. PIF was not effective in cultures of mouse L cells or in cultures of chick embryo fibroblasts. Preliminary data suggest that PIF is heat stable at 85°C for one-half hr, nondialyzable, nonsedimentable at 90,000 × G and is destroyed by trypsin.
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