Oxygen therapy is an essential component of treatment for newborns with respiratory distress, but hyperoxia (>21% O2) is toxic to all cells and causes lung injury. Mechanisms of cytotoxicity from, or tolerance to hyperoxia are not completely understood. A stable mutant line of HeLa cells(called HeLa-80 cells) was selected to grow in 80% O2, yet these cells show no increases in any known antioxidants. Giant 2-D gel electrophoresis was used to compare the patterns of protein synthesis between HeLa-80 cells and the parental cell line (HeLa-20). Out of about 10,00 spots analyzed there were only 15 consistent differences. If any of the proteins are regulated at the mRNA level, it should facilitate the cloning of cDNAs that encode them. Toward that end, we have used the differential display polymerase chain reaction to isolate and identify partial cDNA fragments corresponding to mRNAs that are differentially expressed in HeLa-20 and -80 cells. To date, we have screened approximately 50% of the mRNA complexity of the cells. A total of 68 candidate fragments have been delineated. Northern blots of HeLa-20 and -80 cell mRNA were probed with radiolabeled fragments from 14 of the candidates; 6 of them correspond to differentially-expressed mRNAs. To date, complete DNA sequence analysis of two of these fragments indicates they are derived from novel genes. These genes are expressed at different levels in a wide variety of human tissues. Expression of their full-length cDNAs will be used to determine their potential role in resistance or sensitivity to O2 toxicity.(Funded by grants from the American Heart Association and Winthrop-University Hospital).
(Spon. by Jonathan Davis)