J. gen. ViroL (I977), 37, 221-223

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Repeated ' Superinduction' o f Interferon in H u m a n Diploid Fibroblast Cultures (Accepted I3 June I977) SUMMARY

Human diploid fibroblast cultures induced to make interferon by the combination of polyriboinosinic acid-polyribocytidylic acid, cycloheximide and actinomycin D degenerate thereafter, owing to the irreversible nature of the inhibition induced by actinomycin D. However, cultures superinduced with the DNA-dependent RNA synthesis inhibitor 5,6-dichloro-I-fl-o-ribofuranosylbenzimidazole (DRB) survive, owing to the reversible nature of the inhibition induced by DRB, and can again be superinduced on several occasions. Human fibroblast interferons suitable for clinical trials can be produced from primary human leukocyte suspensions or from human diploid fibroblast cultures. The production of interferons from human fibroblast cultures has been greatly facilitated by the selective use of metabolic inhibitors to enhance interferon production (Vilcek, Rossman & Varacalli, I969; Tan et al. I97o; Tan, Armstrong & Ho, I97I). This process has been generally referred to as "superinduction' and is normally accomplished by combined treatment of cultures with the synthetic double-stranded RNA, polyriboinosinic acid-polyribocytidylic acidpoly(I).poly(C), cycloheximide and actinomycin D (Havell & Vilcek, I972). However, actinomycin D-induced inhibition of DNA-dependent RNA synthesis is irreversible. Thus, after the considerable investment of time and materials required to grow and age human diploid fibroblast cultures to the point and passage-level where they can be induced to produce interferon, the cultures must be discarded after a single superinduction involving actinomycin D. Recently, however, Sehgal, Tamm & Vilcek 0975, I976a, b) have reported that interferon can be superinduced in human diploid fibroblast cultures by a combination of poly(I).poly(C), cycloheximide and 5,6-dichloro-I-fl-D-ribofuranosylbenzimidazole (DRB). An important feature is that the inhibitory effect of this antimetabolite on nuclear heterogeneous (hn) RNA and messenger RNA is readily reversible by washing it from the cultures (Tamm, Hand & Caliguiri, I976), and it is relatively non-toxic for cultured cells (Tamm & Sehgal, I977). We speculated therefore that the superinduction procedures employing DRB, instead of actinomycin D, could be repeatedly performed on the same human fibroblast cultures, so that several yields of interferon could be derived from each cell passage-level. Human skin fibroblast cultures, designated FS-4, were generously provided by Dr E. A. Havell (New York University) and were used between passages I5 and 2o. These cells were inoculated into 6o mm plastic tissue culture plates (5 × IO5 cells/plate) in Eagle's minimal essential medium (MEM) containing Io % foetal calf serum (FCS). Cultures were grown at 37 °C for 7 days before use in initial experiments. Such cultures can be maintained as confluent monolayers for at least 6 weeks by weekly medium changes. DRB was kindly provided by Dr P. Sehgal (Rockefeller University, New York). Cycloheximide and actinomycin D were purchased from Calbiochem (La Jolla, California). When FS-4 cultures were I week old they were divided into four groups: group I

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Table I. Repeated superinduction of interferon in human diploid fibroblast cultures

induction with:

Experimental group*

Poly(i). poly(C)

I 2 3 4

Poly(D.poly(C) + Cycloheximide + Actinomycin D

] 2 3

Poly(I). poly(C) JrCycloheximide +

DRB

Interferon production by cultures induced on day: 28 7 14 2I 300 ND "}" ND ND i oooo ND ND

500 250 ND ND

600 500 I5o ND

200 50O 3¢O 2O0

< to 2oooo ND

ND xo 5ooo

ND ND