Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (149K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (10)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Smith, R. D.
Right arrow Articles by Schechter, A. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smith, R. D.
Right arrow Articles by Schechter, A. N.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2000, Vol. 28, No. 24 4998-5004
© 2000 Oxford University Press

Quantitative analysis of globin gene induction in single human erythroleukemic cells

Reginald D. Smith, James D. Malley and Alan N. Schechter*

Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases and Computational Bioscience and Engineering Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA

The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in {gamma}/( {gamma} + ß) mRNA ratios, with differential effects on ß-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 µM hemin treatment. Hemin exposure increases total hemoglobin levels by ~9-fold and total {alpha}-, {varepsilon}- and {gamma}-globin mRNA levels by 1.5–2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.

* To whom correspondence should be addressed at: Laboratory of Chemical Biology, NIDDK, NIH, Building 10, Room 9N307, 10 Center Drive, MSC 1822, Bethesda, MD 20892-1822, USA. Tel: +1 301 496 5409; Fax: +1 301 402 0101; Email: aschecht{at}helix.nih.gov


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
S. Harju-Baker, F. C. Costa, H. Fedosyuk, R. Neades, and K. R. Peterson
Silencing of A{gamma}-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the -566 GATA Site
Mol. Cell. Biol., May 15, 2008; 28(10): 3101 - 3113.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Sangerman, M. S. Lee, X. Yao, E. Oteng, C.-H. Hsiao, W. Li, S. Zein, S. F. Ofori-Acquah, and B. S. Pace
Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves {gamma}-globin activation by CREB1 and ATF-2
Blood, November 15, 2006; 108(10): 3590 - 3599.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
S. Addya, M. A. Keller, K. Delgrosso, C. M. Ponte, R. Vadigepalli, G. E. Gonye, and S. Surrey
Erythroid-induced commitment of K562 cells results in clusters of differentially expressed genes enriched for specific transcription regulatory elements
Physiol Genomics, September 16, 2004; 19(1): 117 - 130.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
J. Vadolas, H. Wardan, M. Orford, R. Williamson, and P. A. Ioannou
Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin
Hum. Mol. Genet., January 15, 2004; 13(2): 223 - 233.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Vadolas, H. Wardan, M. Orford, L. Voullaire, F. Zaibak, R. Williamson, and P. A. Ioannou
Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human beta -globin locus in erythropoietic cells
Blood, December 1, 2002; 100(12): 4209 - 4216.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.