Ribonucleic acid-induced changes in mammalian cells.

Previous experiments have shown that under proper conditions ribonucleic acid (RNA) can indeed initiate differentiation. " 2 Of particular interest in this work is the development of highly specific structure. That the type of structure developed correlates with the tissue source of RNA indicates a response of the reacting tissue to a message carried directly by RNA or RNA-containing substances of the inducing tissue. Thus, on the cellular level, when a piece of the epidermal ectoderm of urodele gastrula is transplanted into the presumptive kidney area, the ectoderm contributes to the formation of extra kidney tubules3 and develops into muscle in the presumptive muscle area.4 At the molecular level, ribonucleoproteins (RNP) from different tissues were found to be capable of inducing the formation of muscle,', 6 blood,7 thymus,' and other tissues.2 Some two years ago, an examination of the effects of normal RNA on mouse ascites cells was initiated. After incubation with liver RNA, only 10 per cent of the transplanted ascites cells gave rise to tumors. The loss of the tumor-forming ability was correlated with induced cellular changes rather than with regression caused by either cell death or immunological response.8 The cellular changes were shown in vivo by the lack of mitotic division and invasiveness. In vitro, they were demonstrated by the acquired capacity of synthesizing the liver protein, serum albumin.9 In this communication, observations which indicated an inhibitory effect of RNA on tumor growth8' 9 are presented in detail. These studies and comparable studies with the Novikoff hepatoma'0 appear to support the concept that RNA induces cellular transformation in mammalian cells. Materials and Methods. -Mice and ascites cells: During 1959 and 1960, white Swiss mice of the Rockefeller Institute, and in 1961, Swiss Webster mice supplied by Pied Piper Farms were used in this study. The ascites tumor cells were developed and maintained through successive transfers in the peritoneal cavity of the Princeton strain by Dr. John B. Nelson of the Rockefeller Institute, whose generosity in supplying ascites fluid biweekly during 1959 and 1960 the authors are pleased to acknowledge. Upon receipt of the cell suspension, our customary procedure was to wash the ascites cells with 0.9% saline. Intramuscular injection of the washed cells give rise to solid tumors,'I and the mice usually died within a month. For the preparation of tumor RNA, solid tumors without necrosis (8 to 10 days) were used as source material. Mice from the same litter were used for control and experimental series. The number in each series was limited to 5 to 8 mice, weighing 10 to 15 grams each. Isolation of RNA: RNA was prepared from mouse and calf liver (L-RNA) and solid tumor (T-RNA) by a modified Kirby procedure involving low-speed centrifugation.2 Beginning in November 1960, the isolated RNA was centrifuged at 40,000 RPM (Spinco Model-L Rotor #40) for 30 min. The supernatant fluid was subsequently treated with ether and the ether was expelled by nitrogen. The preparations were used immediately or frozen in dry ice-acetone and stored at -20'C for 7 to 10 days. No apparent loss of activity was detected. Ultraviolet absorption spectra of both preparations of RNA were similar, with the lowest absorption at 230 m/u and the highest at 258 miu. In each case, the ratio, absorption at 260 mu/absorption at 230 m,4, exceeded 2.2. The amount of RNA calculated from the orcinol determination, the phosphorus content, and the absorption at 260 myA agreed, thus indicating that the sub-