Elsevier

Gene

Volume 569, Issue 2, 15 September 2015, Pages 182-190
Gene

Review
HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution

https://doi.org/10.1016/j.gene.2015.05.065Get rights and content

Highlights

  • 293 cells have characteristics of neuronal cells and may be of adrenal gland origin.

  • A highly aberrant karyotype makes transcriptome of 293 cells severely deregulated.

  • 293 cells are tumorigenic, forming tumors with varying frequency/size among studies.

  • The diverse cancer-associated genes promote chromosome changes in 293 cells.

  • A procedure of plasmid DNA transfection can impact genotype and phenotype.

Abstract

293 cell line (widely known as the Human Embryonic Kidney 293 cells) and its derivatives were the most used cells after HeLa in cell biology studies and after CHO in biotechnology as a vehicle for the production of adenoviral vaccines and recombinant proteins, for analysis of the neuronal synapse formation, in electrophysiology and neuropharmacology. Despite the historically long-term productive exploitation, the origin, phenotype, karyotype, and tumorigenicity of 293 cells are still debated. 293 cells were considered the kidney epithelial cells or even fibroblasts. However, 293 cells demonstrate no evident tissue-specific gene expression signature and express the markers of renal progenitor cells, neuronal cells and adrenal gland. This complicates efforts to reveal the authentic cell type/tissue of origin. On the other hand, the potential to propagate the highly neurotropic viruses, inducible synaptogenesis, functionality of the endogenous neuron-specific voltage-gated channels, and response to the diverse agonists implicated in neuronal signaling give credibility to consider 293 cells of neuronal lineage phenotype. The compound phenotype of 293 cells can be due to heterogeneous, unstable karyotype. The mean chromosome number and chromosome aberrations differ between 293 cells and derivatives as well as between 293 cells from the different cell banks/labs. 293 cells are tumorigenic, whereas acute changes of expression of the cancer-associated genes aggravate tumorigenicity by promoting chromosome instability. Importantly, the procedure of a stable empty vector transfection can also impact karyotype and phenotype. The discussed issues caution against misinterpretations and pitfalls during the different experimental manipulations with 293 cells.

Introduction

293 cell line (original name designates the number of a successful transformation experiment; more widely known as the Human Embryonic Kidney 293 cells (HEK293)) was derived in 1973 by exposing the human primary embryonic kidney cell culture of an aborted embryo to the mechanically sheared DNA of adenovirus type 5 (AD5) (Graham et al., 1977). A recent analysis showed that 293 cell line and its derivatives (e.g., 293T or 293S) harbor 5–6 copies of AD5 DNA fragments encoding early gene 1 (E1) (Lin et al., 2014), and this copy number did not change from that originally reported (4–5 fragments) (Graham et al., 1977). An AD5 DNA integration site is located in pregnancy-specific beta-1-glycoprotein 4 (PSG4) gene, which was mapped to the chromosome region 19q13.2 (Lin et al., 2014, Louis et al., 1997). 293 cell line and its derivatives have been the most frequently used cells after HeLa in cell biology studies and after CHO in biotechnology (Lin et al., 2014) as a vehicle for the production of adenoviral vaccines (Kovesdi and Hedley, 2010) and recombinant proteins (Geisse and Fux, 2009) as well as for analysis of the neuronal synapse formation in mixed-culture assays (Biederer and Scheiffele, 2007), in electrophysiology and neuropharmacology (Thomas and Smart, 2005, Varghese et al., 2006). Also, 293 cells were often used as a model for studying the transforming/oncogenic properties of cancer-associated genes (Table 1) but sometimes incorrectly supposed as non-tumorigenic or even “normal” human cell line (discussed in Kavsan et al., 2011, Stepanenko and Kavsan, 2012). Despite the widespread and historically long-term productive exploitation in cell biology, biotechnology, and cancer research, an origin, phenotype, karyotype instability, and tumorigenicity of 293 cell line are still disputed and debated.

Section snippets

293 cell line: of kidney or adrenal gland origin and epithelial or neuronal phenotype?

293 cells have been considered the kidney embryonic epithelial cells (e.g., Ashokkumar et al., 2006, Cusick et al., 2010) or even fibroblasts (e.g., Cooper et al., 1998, Yung et al., 2012) and shown to express markers of a kidney developmental stage between the condensed mesenchyme and epithelial S-shaped body but not markers of the differentiated tubular segments, proximal tubules or collecting ducts (Torban and Goodyer, 1998). Moreover, an ectopic expression of transcription factor paired box

The complex karyotype of 293 cells and genome instability-driving mechanisms

293 cells are considered the female origin referring to the presence of several X chromosomes and lack of Y chromosome when conventional cytogenetics is employed (Gilbert et al., 2000, Machida et al., 2009, Meir et al., 2011). Actually, the high-coverage genome sequencing did not reveal a trace of Y-chromosome-derived sequence (Lin et al., 2014). Also, the short tandem repeat analysis with amelogenin marker showed female genotype of 293 cells (American Type Culture Collection, ATCC).

Karyotype

Both stable plasmid DNA transfection and overexpression of cancer-associated genes promote genome-phenotype evolution of tumorigenic 293 cell line

To study the dynamics of the 293 genome under the procedures commonly used in laboratory investigations and in biotechnological engineering, Lin et al. analyzed 293 cell line (from ATCC) and its derivatives using the multiplex fluorescence in situ hybridization, high-coverage genome sequencing, SNP array, and exon array: 1) 293T cell line, which expresses a temperature-sensitive allele of the SV40 T antigen; 2) 293S cell line, which was adapted to growth in suspension; 3) 293SG cell line, which

Concluding remarks

We raise a concern about the use of 293 cell line as a model in studying renal functions or as “normal” human cells in transformation transgenic studies and point out to a significant karyotype diversification of 293 cells and their laboratory/biotechnological derivatives. The aberrant karyotype, genomic heterogeneity, and constitutive expression of adenoviral E1 gene make transcriptome of 293 cells severely deregulated, bringing to naught affords to elucidate an authentic cell type of origin

Conflict of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported in frames of the programs “Fundamental grounds of molecular and cell biotechnologies” №38/14 and “Nanotechnologies and nanomaterials for 2010–2014 years” №5.16.3.14 by the National Academy of Sciences of Ukraine (NASU).

References (154)

  • A. Dereli-Öz et al.

    Studies of genomic copy number changes in human cancers reveal signatures of DNA replication stress

    Mol. Oncol.

    (2011)
  • N. Donnelly et al.

    Dynamic karyotype, dynamic proteome: buffering the effects of aneuploidy

    Biochim. Biophys. Acta

    (2014)
  • P. Duesberg et al.

    Cancer drug resistance: the central role of the karyotype

    Drug Resist. Updat.

    (2007)
  • S. Geisse et al.

    Recombinant protein production by transient gene transfer into mammalian cells

    Methods Enzymol.

    (2009)
  • S.L. Gilbert et al.

    XIST RNA associates with specific regions of the inactive X chromatin

    J. Biol. Chem.

    (2000)
  • B. He et al.

    Human embryonic kidney (HEK293) cells express endogenous voltage-gated sodium currents and Na v 1.7 sodium channels

    Neurosci. Lett.

    (2010)
  • H.H. Heng et al.

    Evolutionary mechanisms and diversity in cancer

    Adv. Cancer Res.

    (2011)
  • M. Jo et al.

    The urokinase receptor promotes cancer metastasis independently of urokinase-type plasminogen activator in mice

    Am. J. Pathol.

    (2009)
  • A. Klein et al.

    Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes

    Cancer Genet. Cytogenet.

    (2010)
  • G.-D. Li et al.

    CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells

    J. Biol. Chem.

    (2008)
  • L. Li et al.

    Cancer-causing karyotypes: chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function

    Cancer Genet. Cytogenet.

    (2009)
  • N. Louis et al.

    Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line

    Virology

    (1997)
  • I.M. Macias-Perez et al.

    Mouse EP3 alpha, beta, and gamma receptor variants reduce tumor cell proliferation and tumorigenesis in vivo

    J. Biol. Chem.

    (2008)
  • S.N. Madhusudana et al.

    Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines

    Int. J. Infect. Dis.

    (2010)
  • B.Y. Abdallah et al.

    Why unstable genomes are incompatible with average profiles single cell heterogeneity

    (2013)
  • A. Adey et al.

    The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line

    Nature

    (2013)
  • S. Akli et al.

    Tumor-specific low molecular weight forms of cyclin E induce genomic instability and resistance to p21, p27, and antiestrogens in breast cancer

    Cancer Res.

    (2004)
  • F. al-Mulla et al.

    RAF kinase inhibitory protein (RKIP) modulates cell cycle kinetics and motility

    Mol. Biosyst.

    (2011)
  • B.A. Arrick et al.

    Altered metabolic and adhesive properties and increased tumorigenesis associated with increased expression of transforming growth factor beta 1

    J. Cell Biol.

    (1992)
  • B. Ashokkumar et al.

    Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms

    Am. J. Physiol. Ren. Physiol.

    (2006)
  • L. Bardwell

    The mutagenic and carcinogenic effects of gene transfer

    Mutagenesis

    (1989)
  • T. Biederer et al.

    Mixed-culture assays for analyzing neuronal synapse formation

    Nat. Protoc.

    (2007)
  • L. Bylund et al.

    Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches

    Cytogenet. Genome Res.

    (2004)
  • S.A. Campbell et al.

    Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells

    J. Virol.

    (2005)
  • M. Canis et al.

    CD133 induces tumour-initiating properties in HEK293 cells

    Tumour Biol.

    (2013)
  • R. Capper et al.

    The nature of telomere fusion and a definition of the critical telomere length in human cells

    Genes Dev.

    (2007)
  • F. Castiglioni et al.

    Role of exon-16-deleted HER2 in breast carcinomas

    Endocr. Relat. Cancer

    (2006)
  • Y.-H. Chang et al.

    Epstein–Barr virus BGLF4 kinase retards cellular S-phase progression and induces chromosomal abnormality

    PLoS One

    (2012)
  • C. Chao et al.

    Constitutively active CCK2 receptor splice variant increases Src-dependent HIF-1 alpha expression and tumor growth

    Oncogene

    (2007)
  • J.D. Cheng et al.

    Promotion of tumor growth by murine fibroblast activation protein, a serine protease, in an animal model

    Cancer Res.

    (2002)
  • J.D. Cheng et al.

    Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth

    Mol. Cancer Ther.

    (2005)
  • J.K. Cooper et al.

    Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture

    Hum. Mol. Genet.

    (1998)
  • D. Corcos

    Unbalanced replication as a major source of genetic instability in cancer cells

    Am. J. Blood Res.

    (2012)
  • K. Crasta et al.

    DNA breaks and chromosome pulverization from errors in mitosis

    Nature

    (2012)
  • F. Depontieu et al.

    Loss of endocan tumorigenic properties after alternative splicing of exon 2

    BMC Cancer

    (2008)
  • P. Duesberg et al.

    Immortality of cancers: a consequence of inherent karyotypic variations and selections for autonomy

    Cell Cycle

    (2013)
  • P. Duesberg et al.

    The chromosomal basis of cancer

    Cell. Oncol.

    (2005)
  • P. Duesberg et al.

    Is carcinogenesis a form of speciation?

    Cell Cycle

    (2011)
  • P. Duesberg et al.

    Origin of metastases: subspecies of cancers generated by intrinsic karyotypic variations

    Cell Cycle

    (2012)
  • D. Foudah et al.

    Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

    Cell. Mol. Biol. Lett.

    (2013)
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