Bypass of senescence, immortalization, and transformation of human hematopoietic progenitor cells.
We attempted to extend the lifespan of CD34+ stem/progenitor cells in human cord blood (CB) by transduction with lentiviral vectors carrying the human telomerase catalytic subunit (hTERT) and/or the human papillomavirus type 16 (HPV16) E6 and E7 oncogenes. We found that hTERT was incapable of prolonging the replicative capacity of CB cells maintained under serum-free conditions in the presence of stem cell factor, Flt3 ligand, thrombopoietin, and interleukin-3 beyond 4 months (n=3). However, transduced CB cells cultured in the same cytokine cocktail constitutively expressing HPV16 E6/E7 alone (n=2) or in concert with hTERT (n=9) continued to proliferate, giving rise to permanent (>2 years) cell lines with a CD45+ CD34- CD133+/- CD44+ CD235a+ CD71+ CD203+ CD33+ CD13+ myeloerythroid/mast cell progenitor phenotype. Notably, CB cell cultures expressing only HPV16 E6/E7 went through a crisis period, and the resulting oligoclonal cell lines were highly aneuploid. By comparison, the CB cell lines obtained by coexpression of HPV16 E6/E7 plus hTERT exhibited near-diploid karyotypes with minimal chromosomal aberrations, concomitant with stabilization of telomere length, yet were clonally derived. The immortalized E6/E7 plus hTERT-expressing CB cells were not tumorigenic when injected intravenously or subcutaneously into sublethally irradiated immunodeficient nonobese diabetic/severe combined immunodeficient mice but could be converted to a malignant state by ectopic expression of a v-H-ras or BCR-ABL oncogene. These findings provide new insights into the mechanisms governing the senescence checkpoint of primitive human hematopoietic precursors and establish a paradigm for studies of the multistep process of human leukemogenesis.
Akimov
Sergey S
SS
Department of Anatomy and Cell Biology, The George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037, USA.
Ramezani
Ali
A
Hawley
Teresa S
TS
Hawley
Robert G
RG
D018952:Antigens, CD34 / Q000096:biosynthesis; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics / Q000502:physiology*; D016922:Cellular Senescence; D004268:DNA-Binding Proteins / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000502:physiology / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000502:physiology / Q000821:virology; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D018952:Antigens, CD34; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D019098:Telomerase