Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures.
Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am-MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.
Rothstein
L
L
Pierce
J H
JH
Klassen
V
V
Greenberger
J S
JS
D000818:Animals; D001402:B-Lymphocytes; D001853:Bone Marrow; D002448:Cell Adhesion; D002472:Cell Transformation, Viral; D003114:Colony-Forming Units Assay; D046508:Culture Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D004854:Herpesvirus 4, Human; D006801:Humans; D009052:Leukemia Virus, Murine; D051379:Mice; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012190:Retroviridae; D009053:Sarcoma Viruses, Murine; D014779:Virus Replication
D012194:RNA-Directed DNA Polymerase