With the long-term goals of (1) analyzing transforming genes in hemopoietic cells, (2) defining unknown functions of cellular genes, (3) studying globin gene regulation, and (4) examining the potential of genetic therapy, we have examined the effects of a cellular oncogene on hemopoietic cells in vitro. A virus chimera (EJ-1 virus) was constructed by ligating portions of Moloney leukemia virus and EJ-l, the transforming principle (encoding a ras p2l) cloned from human bladder carcinoma cells. DNA of this replication-defective EJ-1 virus clone was transfected into psi-2 helper cells and transformed foci picked for expansion. Media was harvested from these expanded cultures and tested for transforming activity on 3T3 cells and for ras-induced in vitro erythroid transformation. 3T3 cells were non-productively transformed since the culture media were negative for transforming activity, XC and reverse transcriptase. The p21 encoded by EJ-1 was not phosphorylated and migrated as a single band on polyacrylamide gel electrophoresis (PAGE). Thus, the EJ-1 protein could be distinguished from the p21 doublet observed in Harvey sarcoma virus-infected cells. EJ-1 infection of murine hemopoietic cells induced large erythroid colonies (containing hemoglobin-positive and hemoglobin-negative cells) which appeared at 6-10 days post-infection. Picked erythroid cells were found positive for p21 by immunofluorescence. We have previously noted a variety of phenotypes of erythroid colonies induced by Friend, Harvey, Abelson, Rous and other transforming viruses. Evaluation of phenotypic characteristics of EJ-1 induced colonies with respect to time course, erythropoietin sensitivity, proportion of differentiated cells, and replating potential, most closely resembled that of erythroid colonies induced by other ras-containing viruses.
NIH ExPorter
NIH ExPorter