Summary
The flow cytometric detection of fluorescent in situ hybridization (FISH) performed on intact cells in suspension is a recently described method (Bauman et al. 1989). We studied the application of this method for monitoring cellular differentiation. The amount of rRNA which is taken for a good indicator of growth in size, the rate of protein synthesis and the G0–G1 transition was followed by FISH. For this purpose biotinylated single stranded RNA probes obtained by transcription from a 2.1 kb BglII-EcoRI fragment of the human 28S ribosomal RNA gene subcloned into plasmid pGEM2 were used. K-562 leukaemic cells, used as targets, were induced to differentiate by dimethyl sulfoxide, phorbol myristate acetate and hemin. In the last two cases the cell cycle analysis, growth kinetics, cellular morphology and immunophenotyping indicated differentiation into monocytic and erythroid direction, respectively. The differentiation was accompanied by a rapid increase followed by a decrease to the base level of rRNA. This was not observed in the uninduced exponentially growing control cells. Based on our results, we propose that the FC-FISH detection of the rRNA level is a valuable method to distinguish between cell subpopulations. We propose that using other probes, FC-FISH will become useful to monitor different cellular processes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baserga R (1984) Growth in size and cell DNA replication. Exp Cell Res 151:1–5
Bauman JGJ, Bentvelzen P (1988) Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization. Cytometry 9:517–524
Bauman JGJ, Pinkel D, Trask BJ, Ploeg M van der (1989) Flow cytometric measurement of specific DNA and RNA sequences. In: Gray IW (ed) Flow cytogenetics. Academic Press, San Diego, pp 276–296
Bayer JA, Bauman JGJ (1990) Flow cytometric detection of β-globin mRNA in murine haemopoetic tissues using fluorescent in situ hybridization. Cytometry 11:132–143
Borelli TJ, Miyamoto S, Wertheimer SJ, Wu JM (1987) Changes in thymidine kinase activity during differentiation of HL-60 leukemic cells. Leuk Res 11:469–473
Cox KH, Deleon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymetric RNA probes. Dev Biol 101:458–502
Darnell JE, Lodish HF, Baltimore D (1986) Analysing pulse labeled RNA in cell fractions: The discovery of RNA processing. In: Darnell JE, Lodish HF, Baltimore D (eds) Molecular cell biology. Scientific American Books, New York, p 308
Engh GJ van den, Stokdijk W (1989) Parallel processing data aquisition system for multilaser flow cytometry and cell sorting. Cytometry 10:282–292
Erikson JM, Rushford CL, Dorney DJ, Wilson GN, Schmickel RD (1981) Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments. Gene 16:1–9
Fukuda M (1981) Tumor-promoting phorbol diester-induced specific changes in cell surface glycoprotein profile of K-562 human leukemic cells. Cancer Res 41:4621–4628
Gewirtz AM, Burger D, Rado TA, Benz EJ Jr, Hoffman R (1982) Constituve expression of platelet glycoproteins by the human leukemia cell line K-562. Blood 60:785–789
Jonker R, van Rotterdam A, Bauman J, Visser J (1987) Analysis of eight parameter list mode data and parameter estimation of uni- and bivariate distributions using a Kalman curve fitting procedure. Cytometry [Suppl] 1:98
Kohno H, Tsketani S, Tokunaga R (1986) Phorbolester-induced regulation of transferrin receptors in human leukemia K-562 cells. Cell Struc Funct 11:181–190
Krumlauf R, Jeanpierre M, Young BD (1982) Construction and characterization of genomic liberies from specific human chromosomes. Proc Natl Acad Sci USA 79:2971–2975
Lozzio CB, Lozzio BB (1975) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321–334
Murnane MJ, Phipps PM, Denecamp DS (1988) Expression and induction of cathepsin B and D in K-562 cells. Exp Hematol 16:33–37
Pellici PG, Testa U, Thomopoulos P, Tabilio A, Vainchenker W, Titeux M, Gourdin MF, Rochant H (1984) Inhibition of transferrin binding and iron uptake of hematopoietic cell lines by phorbol esters. Leuk Res 8:597–609
Rovera J, Ferrero D, Pagliardi GL, Vartikar J, Pessano S, Bottero L, Abraham S, Lebman D (1982) Induction of differentiation of human myeloid leukemias by phorbol diesters: phenotypic changes and mode of action. Ann NY Acad Sci 397:211–220
Stephen HC, Cooper RA (1980) Decreased membrane fluidity during differentiation of human promyelocytic leukemic cells in culture. Blood 56:227–232
Tettero PAT, Massaro F, Mulder A, Screuder-van Gelder, Borne EAG von dem (1984) Megakaryoblastic differentiation of proerythroblastic K-562 cell line cells. Leuk Res 8:197–206
Trask B, Engh G van den, Landegent J, Jansen Wal N in de, Ploeg M van der (1985) Detection of RNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230:1401–1403
Trask B, Engh G van den, Pinkel D, Mullikin J, Dekken H van, Grey J (1988) Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization. Hum Genet 78:251–259
Wanda PE, Lee LT, Howe C (1981) A spectrophotometric method for measuring hemoglobin in erythroleukemic cells (K-562). J Histochem Cytochem 29:1442–1444
Wyse JW, Butterfield DR (1989) Interaction of hemin with erythrocyte membranes: alterations in the physical state of the major sialo-glycoprotein. Biochim Biophys Acta 979:121–126
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pajor, L., Bauman, J.G.J. Flow cytometric measurement of rRNA levels detected by fluorescent in situ hybridization in differentiating K-562 cells. Histochemistry 96, 73–81 (1991). https://doi.org/10.1007/BF00266764
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00266764