SHORT DNA-SEQUENCES FROM THE CYTOPLASM OF MOUSE-TUMOR CELLS INDUCE IMMORTALIZATION OF HUMAN-LYMPHOCYTES IN-VITRO

Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA se quences that induce unlimited proliferation (''immortalization'') of h uman lymphocytes after transfection in vitro. By equilibrium centrifug ation of cytoplasmic lysates in a neutral CsCl gradient, the immortali zing activity was recovered together with extramitochondrial fractions at high salt densities (1.85-1.87 g/cm3). Unexpectedly, these fractio ns contain linear DNA molecules of 50-500 bp in length. In contrast, c ytoplasts of primary, senescent cells (mouse embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding fractions. Cytopl asmic DNA isolated from high-density fractions of mouse tumor cells wa s cloned in subset libraries, and of 45 DNA sequences we identified 2 clones-one from L929 cytoplasts (203 bp) and another one from the cyto plasm of Ehrlich ascites cells (372 bp)-that induce unlimited prolifer ation of human lymphocytes in vitro. Immortalized lymphoid cells harbo r 1-5 copies of transfected DNA integrated into chromosomal DNA, where as about 100 copies were found as episomal DNA in the cytoplasmic frac tion. No immortalization could be induced by transfection of nuclear D NA randomly fragmented to 200-500 bp. Although the cloned DNA sediment ed at 1.70 g/cm3, after transient transfection into lymphocytes, these DNA sequences form salt-stable complexes that sediment in fractions a t the same high density (1.82-1.88 g/cm3) from which they were origina lly cloned. The high-density banding of these cytoplasmic DNA sequence s may be due to association with RNA and/or with (metallo-) proteins i n vivo. Since both cloned DNA sequences with immortalizing activity ha ve stop codons for protein translation in an possible reading frames, immortalization may be induced by insertional inactivation or function al suppression of genes that are needed to be expressed during cellula r senescence or programmed cell death.