Preferential Inhibition of the Growth of Virus-Transformed Cells in Culture by Rifazone-8 ₂ , a New Rifamycin Derivative

Abstract

Rifampicin, a well-known derivative of rifamycin, has been shown to inhibit the replication of vaccinia virus in tissue culture when added at very high levels (>100 ug/ml) (15,16) and to reduce the incidence of adenovirus-induced tumors in male hamsters (16). In chick cells it has been argued that rifampicin (a) inhibits focus formation by Rous sarcoma virus (RSV) (1), (b) has no effect on transformation, as it is toxic to both normal and transformed cells (17), and (c) is preferentially toxic to transformed cells (20). Variation in culture conditions, serum concentration, and cell density undoubtedly play a role in such contradictory findings. For example, whether or not amphotericin B is present in culture medium may drastically change the result of focus inhibition (4, 19). Furthermore, the high concentration of rifampicin used in these experiments (20-80 ug/ml) under some conditions is quite toxic to normal cells and makes interpretation of these data very difficult. Rifampicin itself has little or no effect on activity of RNA-directed DNA polymerase in vitro at concentrations used in tissue culture studies (7,17,20). We have tested several new rifamycin derivatives synthesized in this laboratory that have been shown to be inhibitors of RNA-directed DNA polymerase in vitro and Abbreviations: R-82 or rifazone-82, rifaldehyde-N,N-di-n-octyl hydrazine-hydrazone; RSV, Rous sarcoma virus; B-RSV, Bryanhigh-titer strain of RSV; SR-RSV, Schmidt-Ruppin subgroup A strain of RSV; Rif, Rifampicin; DMB, 2',6'-dimethyl4'-benzyl-4'-desmethylrifampicin; (Me)2SO, dimethyl sulfoxide.inhibitors of focus formation in vivo in other cell systems (4-6).In addition, we have tested rifazone-82 (R-82), a new rifamycin which is to date the most selective inhibitor of viral RNAdirected DNA polymerase in vitro (6, 21). We find that at low concentrations (3-10 gg,ml), R-82 can selectively inhibit the growth of transformed cells and prevent focus formation while allowing the normal cell growth and function to continue. MATERIALS AND METHODSGrowth of Cell Cultures. Primary cultures were prepared from 10-day-old C/O or C/B type SPF chick embryos as described (23,24), except that amphotericin B (Fungizone) was eliminated at this point (25). The cells were seeded in medium 199, which was supplemented with tryptose phosphate broth (2%), calf serum (1%), and heated chicken serum (1%). The medium was changed on day 3. Secondary cultures were prepared 4 days after the primary seeding by trypsinization of primary cultures and were seeded at the desired cell concentration in 60-or 35-mm petri dishes. An additional 1 mg/ml of glucose was added to the medium at this time, bringing the final concentration of glucose to 11.00 mM, and calf-serum concentration was raised to 2%. For studies with transformed cultures, half the cells of a single embryo were infected 4 hr after primary seeding with 4 X 105 focusforming units of SR-RSV or B-RSV. Secondary cultures were prepared as above.Focus Assay. Assays were performed essentially as described (22), with sl...

Ascorbic acid inhibits replication and infectivity of avian RNA tumor virus.

“…PAT cells and chicken embryo fibroblasts (CEF) were prepared from 16-and 10-day-old chicken embryos, respectively, as described (4,5). Cells were infected with various strains of Rous sarcoma viruses (6,7), including a temperature-sensitive mutant virus, LA-24 (Prague, Subgroup A), with a defect in the src gene (8). Focus-forming units and infectious centers were determined according to Rubin (9).…”

Detection of an early surface change during oncogenic transformation.

“…Chicken embryo fibroblasts were prepared and cultured as described (8). Primary cultures were infected with virus (LA24) by addition of 3.5 X 106 focus-forming units to approximately 5 X 105 cells in each 100-mm culture dish.…”

Inhibition of Adenocarcinoma TA3 Ascites Tumor Growth by Rifamycin Derivatives

“…Rifa mycin derivatives have also been shown in cell culture studies to be selectively toxic to virally-transformed chick cells [16,17] and leukemic leukocytes [18,19], and to lead to the pro duction of non-infectious progeny virus in transformed chick cells [20], In vivo experimental programs to evaluate the anti tumor potential of rifamycin derivatives have been less exten sive. Studies to date have demonstrated that Rif inhibits Wal ker 256 ascites carcinosarcoma in rats [21] and exerts a pro phylactic effect against adenovirus-induced tumors in male hamsters [22], The derivative DMB has been observed to inhi bit the onset and to decrease the total incidence of carcinogeninduced rat tumors [23].…”