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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 10, 2002 as doi:10.1096/fj.01-0995fje. |
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University of Florida College of Medicine, Department of Neuroscience, McKnight Brain Institute, Gainesville, Florida, USA; and
* Departments of Biology and Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8S 4K1
2Correspondence: E-mail: shaw{at}mbi.ufl.edu
SPECIFIC AIMS
We observed that human embryonic kidney 293 (HEK293) cells (originally isolated from primary human embryo kidney cells transformed by sheared adenovirus 5 DNA) stain strongly and in a clearly filamentous manner with antibody to the major neurofilament (NF) subunit NF-M normally expressed only in neuronal cells. Because this was an unexpected result for a kidney-derived cell line, we screened several other adenovirus as well as SV40 transformed cell lines for expression of several NF proteins. We used microarray analysis to obtain a catalog of the genes expressed by the widely used 293 cell line.
PRINCIPAL FINDINGS
Human 293 cells were found to stain strongly and specifically with a monoclonal antibody to NF-M (Fig. 1
A). This observation was reproduced with several other well-characterized NF-M antibodies and with 293 cells from several sources, including very early passage cells. Double-label immunocytochemistry showed that 293 cells also express large amounts of NF-L, smaller amounts of the NF subunit 
-internexin, and some NF-H as shown by immunocytochemistry and immunoblotting. Figure 1B-F
shows 293 cells labeled with NF-M (red channel) and counterstained (green channel) with various antibodies. NF-L is shown in Fig. 1B
, where exact coexpression gives an orange image. -Internexin is shown in Fig. 1C
, where weaker expression of -internexin gives an orange signal in regions rich in filament proteins. NF-H (Fig. 1D
) shows strong expression in a subpopulation of NF-positive cells. The level of expression in individual cells for NF proteins vs. vimentin (Fig. 1E
) and the keratins (Fig. 1F
) was variable, so that double-label immunocytochemistry reveals orange cells and cells predominantly green and predominantly red. 293 cells stained with antibody to keratin 8 but were negative for desmin, peripherin, and glial fibrillary acidic protein. These findings were confirmed by appropriate immunoblotting experiments, which showed strong signals for NF-L, NF-M, vimentin, keratin 8, and keratin 18 and a weaker signal for -internexin. This general pattern of intermediate filament protein expression is similar to that seen in PC12 cells and Ntera-2 cells, two widely used neuronal lineage cell lines. Several other cell lines derived from human embryonic kidney and retinal cell cultures by adenovirus (Ad) transformation expressed NF proteins in the same fashion as 293 cells. MH12 cells (derived by Ad 5 transformation of HEK cells) and human embryonic retina 224 (HER224) cells, derived by Ad5 transformation of human embryonic retina cultures, showed strong staining with NF antibodies in a manner similar to that seen with 293 cells. We tested two Ad12 transformed cell lines derived from rodent kidney that expressed NF whereas four transformed by Ad5 did not. SV40 transformed cell lines derived from HEK cultures did not express NF proteins, revealing instead only keratin and/or vimentin, as expected for typical epithelial and/or mesenchymal cells. In line with this, cells in primary cultures of human embryonic kidney were positive for keratins and/or vimentin but lacked NF staining.
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Microarray analysis of HEK 293 cells confirmed the presence of mRNA encoding NF-L, NF-M, -internexin, vimentin, keratins 8, and keratin 18 and revealed expression of mRNAs specific for numerous other genes normally expressed in neuronal lineage cells (Table 1)
. The presence of certain of the corresponding proteins was confirmed immunocytochemically. For example, we detected abundant mRNA encoding the serotonin transporter, and the cells stained strongly in a clearly vesicular fashion with serotonin antibody. Similarly, we detected mRNA encoding CGRP-related peptide and neurogranin and observed vesicular staining for proteins with appropriate antibodies. Many genes reported in other microarray studies to be abundantly expressed in neuronal and hematopoietic stem cells were also abundantly expressed in 293 cells.
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CONCLUSIONS AND SIGNIFICANCE
Ad5 transformed 293 cells and several other Ad transformed cells derived from cultures of human embryonic kidney reveal a pattern of intermediate filament expression similar to that seen in early differentiating neurons, which typically express vimentin, -internexin, NF-M, and NF-L, with a small amount of NF-H. In contrast, cells lines derived from primary HEK cell cultures by SV40 transformation lack NF subunits but express keratins and/or vimentin, as expected for cells derived from typical kidney epithelial or mesenchymal cells. To account for the independent isolation of several Ad transformed cell lines from primary human embryonic kidney cultures that all express NF subunits, we propose that neuronal cells are a preferred target for Ad transformation. The presence of a small number of cells with neuronal properties in developing kidney has been reported. These may be the cell type of origin of 293 and other Ad transformed cell lines. That only a small fraction of cells in HEK cultures are susceptible to Ad transformation is consistent with the extreme difficulty in establishing the original 293 cell line and other Ad transformed HEK cells. In contrast to transformation of HEK cells, Ad transformed cell lines are readily obtained from embryonic retinal cell cultures, which contain predominantly neuronal lineage cells. An alternate hypothesis is that Ads reprogram the transcriptional machinery of cells to produce a neuronal phenotype, but this seems less likely. 293 cells are one of the most widely used human cell lines and the fact that they have properties of neuronal lineage cells and not more typical kidney cells could have significant implications for experiments that use these cells as kidney cell controls or as non-neuronal control cells. More important, that Ads preferentially transform cells of neuronal lineage may have implications for mechanisms of Ad-mediated transformation and the role of Ads in tumor induction.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0995fje; to cite this article, use FASEB J. (April 10, 2002) 10.1096/fj.01-0995fje 
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