Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content.
Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100-300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35-40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G1 and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G1 into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G2 + M-phase cells; (2) there are two distinct subpopulations in G1, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G1-phase cells with low nuclear RNA have distinctly longer residence times in G1 compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.
Staiano-Coico
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L
Department of Surgery, Cornell University Medical College, New York 10021.
Darzynkiewicz
Z
Z
McMahon
C K
CK
D000165:Acridine Orange; D002451:Cell Compartmentation / Q000235:genetics*; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D003703:Demecolcine; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis; D015138:RNA, Nuclear / Q000032:analysis*
D012333:RNA, Messenger; D015138:RNA, Nuclear; D012313:RNA; D004247:DNA; D000165:Acridine Orange; D003703:Demecolcine