Lasting changes in the properties of cultured diploid human cells may be produced by a number of factors. Oncogenic viruses "transform" cells, with associated karyotypic changes and the appearance of a variety of new phenotypic characters; and rare spontaneous transformations may produce a similar spectrum of changes.", 2 The gradual "senescence" of serially passaged diploid human cells is evidenced in a progressively slower growth rate, late cytologic and karyotypic changes, and ultimate death.3 Often unrecognized contamination with mycoplasma may also cause changes in metabolic activity. In addition, compaction of growing cells in a monolayer is usually accompanied by a reversible inhibition of motion, macromolecular synthesis, and growth.2 4 Possibly related to the latter effects, certain enzymatic activities5 and specialized biochemical functions6 are markedly influenced by population density.The present communication describes far-reaching and stable changes in the pattern of RNA synthesis which occurred regularly in human diploid cell strains serially propogated in this laboratory, and which were apparently unrelated to any of the foregoing factors.Methods.-Cultures: The cell strains used were all human diploid fibroblasts, five deriving from fetal tissues (adrenal, bone, conjunctiva, skin, and thymus) and three from juvenile skin, one normal (F 1), one galactosemic (Detroit 510), and one hyperglycinemic (BAL). Their source and methods for propagation have been described elsewhere.' Periodic tests of these strains for mycoplasma contaminations in particular after the change in the pattern of RNA synthesis as here described, were generally negative. Cultures were maintained in the presence of penicillin, streptomycin, and aureomycin (5,5, and 10 y/ml, respectively); and tests for mycoplasma were carried out after at least two subcultures in aureomycin-free medium. In nine attempted cultivations of eight different altered cell lines, positive isolations were reported in one. (Three positive results in a single shipment of bacteriologically contaminated specimens are not here included.) Electron microscope examination by Dr. Elliott Robbins of this institution showed no evidence of mycoplasma.Karyotypic examinations of these lines, generously carried out by Dr. Harold P. Klinger, showed them to be euploid after the altered pattern of RNA synthesis to be described had been established.Analysis of cellular RNA: (1) Pulse-labeling and preparation of extracts: Monolayer cultures were pulse-labeled with C'4-uridine (Schwarz BioResearch, Inc., 24-39 jc/millimole, used at 0.05-0.20 ,uc/ml; carrier uridine at 0.008 mM was sometimes added). Incorporation was stopped at appropriate times by decanting the pulsing medium, and washing the cell sheet twice with cold Hanks' salt solution. Nuclear and cytoplasmic fractions were prepared by Dounce homogenization of cells harvested with crystalline trypsin and trypsin inhibitor at 0°as previously described.4, 8 Cytoplasmic fractions were analyzed by sucrose density centrifuga...
