INTERNUCLEOSOMAL DNA FRAGMENTATION IN CULTURED-CELLS UNDER CONDITIONSREPORTED TO INDUCE APOPTOSIS MAY BE CAUSED BY MYCOPLASMA ENDONUCLEASES

DNA fragmentation is a common biochemical hallmark of apoptosis, It is catalyzed by endogenous Ca2+, Mg2+-dependent endonuclease(s). Althoug h the exact identity of the apoptotic endonuclease is still a matter o f debate, a number of candidate nucleases have been proposed like NUC1 8, DNase II and I)Nase I, Relatively large amounts of nucleases are al so expressed by mycoplasmas, cell wall-less bacteria of the class Moll icutes, which are found as contaminants in up to 45 % of the continuou s cell lines in current use, In order to clarify the effect of these p athogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expressio n) and morphological changes characteristic of apoptosis (cell shrinka ge, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis- free and -infected cultures of the human pancreatic adenocarcinoma cel l line PaTu 8902 and of mouse NIH 3T3 fibroblasts, For that purpose me employed cells cultured under standard conditions and cells exposed t o the protein synthesis inhibitor cycloheximide, which is known to ind uce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA deg radation. In contrast, nuclease activities in the molecular range of 4 7 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expressi on of the nucleases and the cycloheximide-induced DNA fragmentation we re suppressed by the prokaryote-specific protein synthesis inhibitor c hloramphenicol. Moreover, partially purified nucleases from supernatan ts of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems ran also be caused by mycoplasmal n ucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internu cleosomal DNA fragmentation in established cell lines has to be regard ed with care, unless mycoplasmal infection can be excluded, or the exi stence of endogenous endonucleases ran be proven, The presence of endo nucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restri cted to infected cells; in both mycoplasma-free and -contaminated cult ures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342, Electron microscopic studies revea led that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells, Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis a s indicated by the parallel occurrence of morphological characteristic s of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).