Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.
We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.
Jensen
U B
UB
Institute of Human Genetics, University of Aarhus, Denmark. uffe@humgen.au.dk
Petersen
M S
MS
Lund
T B
TB
Jensen
T G
TG
Bolund
L
L
D002448:Cell Adhesion; D002453:Cell Cycle; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D003094:Collagen; D003114:Colony-Forming Units Assay; D004261:DNA Replication; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D004854:Herpesvirus 4, Human / Q000235:genetics; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D008081:Liposomes; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D003094:Collagen