The
ideal characteristics of an in vitro
whole-cell model include: equivalence to in
vivo physiology, genotypic and karyotypic stability, high proliferative
capability, and the ability to be used at high passage. Does such a cell type
exist? In this month’s blog, I will discuss human telomerase
(hTERT)-immortalized cells (ICs), which possess all of these qualities1.
hTERT
ICs are derived from primary cells that are made to constitutively express the
catalytic component of the enzyme hTERT. The expression of hTERT allows a cell
to escape the replicative senescence that occurs in primary cells after a
certain number of population doublings (i.e. the ‘Hayflick limit’)2. This limit of proliferation is a
consequence of the fact that each time a cell undergoes mitosis, its
chromosomes shorten by 30 to 200 nucleotides. Telomeres, which are 8,000 nucleotide
base pair noncoding repeat regions that cap either end of a chromosome, prevent
the loss of coding DNA. Thus, an adult mammalian somatic cell may undergo
between 30 to 50 cell divisions before it has lost its telomeres. hTERT is
typically expressed in germ cells, fetal tissue, and some tumor cells, where it
functions to protect mammalian reproductive capability, early development, and
tumor growth, respectively3.
With
the discovery of its telomere-extending properties, it was inevitable that
someone would test hTERT’s ability to extend the lifetime of non-transformed
cultured cells. Interestingly, the stable transfection of telomerase into somatic
human cells has been observed to increase their lifespan 5-fold3. Moreover, primary cells that are
induced to stably express hTERT retain many of their in vivo biologies at high passage, are diploid or near-diploid, and
show gene expression similar to the parental cell1. Furthermore, because hTERT ICs are derived from a single
clone, there is limited lot-to-lot variability, which reduces inconsistencies
among experiments.
hTERT
ICs reveal their usefulness as controls in experiments designed to determine
the pathogenesis of cancer. For example, hTERT-immortalized human bronchial
epithelial cells (HBEC3-KT) have been implemented as an analog for normal cells
in lung cancer studies because of their similarity to primary cells; they do
not exhibit the hallmarks of cancer cells, such as forming colonies in soft
agar or producing tumors when injected into mice. Also, because they are not
transformed, HBEC3-KT are wild type for the tumor suppressor protein p53 and
the oncogene KRASV12. Thus, HBEC3-KT have been used in experiments
as controls against tumor cell lines with those common mutations. These studies
have identified some of the pre-mRNA splicing regulators, such as heterogeneous
nuclear ribonucleoprotein L and splicing regulator p30a, which are responsible for
driving multidrug resistance in non-small cell lung carcinoma cells4,5.
hTERT-ICs
have been used to study the intracellular pathways that drive oncogenesis in
other tissue types as well. For instance, human mammary epithelial cells
(hTERT-HME1) have been utilized as controls in breast cancer studies. Like
HBEC-3KT, hTERT-HME1 present with an untransformed phenotype, and have normal
expression pattern of tumor suppressors and oncogenes. The studies utilizing
hTERT-HME1 as controls have uncovered critical information in the pathogenesis
of breast cancer, such as the sensitization capacity of BRMS1 gene in
ATP-induced growth inhibition and apoptosis, as well as the role of various
caveolin 1 mutations in breast cancer tumorigenesis6,7.
While
hTERT ICs provide researchers with a continuously proliferating source of
physiological controls for their experiments, they also indicate their
usefulness in generating 3D models of complex tissue. The ability to develop 3D
culture models is a growing area for studying drug efficacy, toxicology of therapeutics,
and organogenesis. These culture models enhance target validation studies and
reduce the number of animals needed in toxicological studies. In one study
performed by ATCC scientists, early and late passage co-cultures of
hTERT-immortalized keratinocytes (Ker-CT) and hTERT-immortalized mesenchymal
stem cells (MSCs) formed epidermis-like stratified structures that presented
strata-appropriate markers of differentiation (such as keratin 1, keratin 14,
and filaggrin). In addition, the Ker-CT and hTERT-MSC co-cultures, after
undergoing a scratch test assay, displayed re-epidermalization of their wounds8.
hTERT
IC 3D culture models are not confined to the epidermis; in another example of
the ability of these cells to form physiological structures, hTERT-immortalized
aortic endothelial cells co-cultured with hTERT MSCs formed tubular networks
reminiscent of blood vessels. This variant of the co-culture model also
demonstrated physiological responses to pro- and anti-angiogenic factors. ATCC
scientists observed that vascular endothelial growth factor (VEGF)
concentration-dependently stimulated vascular tubule formation. Interestingly,
the formation of these tubules was inhibited by treatment with VEGF antibodies
(unpublished) or suramin9.
Thus, the fact that hTERT IC-based 3D culture systems show real tissue
responses to physiological stimuli uncovers their potential utility in
pharmaceutical testing and toxicology studies. (Note: If you are interested in
seeing how these studies were performed you can view the epidermal and
angiogenic poster presentations on the ATCC website. ATCC also has numerous hTERT-related
materials, such as the hTERT Cell Culture Guide, recorded Webinar Presentations, and application notes, which can be
found in the ATCC Cell Biology Learning Center.)
Overall,
hTERT ICs possess the flexibility to be implemented as normal controls in
genetic alterations studies, or create organotypic co-culture models. These
cells exhibit the ideal qualities of a whole-cell model, including equivalence
to normal physiology, genotypic and karyotypic stability, high proliferative
capability, and the ability to use at high passage.
References
1. Dickson MA, et al. Human keratinocytes that express
hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span
become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20(4):1436-47, 2000.
2. Shay JW, Wright WE.
Hayflick, his limit, and cellular ageing. Nat
Rev Mol Cell Biol 1(1):72-6, 2000.
3. Bodnar AG, et al. Extension of life-span by
introduction of telomerase into normal human cells. Science 279(5349):349-52, 1998.
4. Goehe RW, et al. hnRNP L regulates the tumorigenic
capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing. J Clin Invest 120(11):3923-39, 2010.
5. Shultz JC, et al. Alternative splicing of caspase 9
is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation
of SRp30a. Cancer Res 70(22):9185-96;
2010.
6. Zhang Y, et al. BRMS1 Sensitizes Breast Cancer
Cells to ATP-Induced Growth Suppression. Biores
Open Access 2(2):77-83, 2013.
7. Lee H, et al. Caveolin-1 mutations (P132L and
null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a
dominant-negative manner and caveolin-1 (-/-) null mice show mammary epithelial
cell hyperplasia. Am J Pathol 161(4):1357-69,
2002.
8. Briley A, et al. Characterization of a
three-dimensional (3D) organotypic skin model using keratinocytes and
mesenchymal stem cells immortalized by hTERT. Poster presented at the annual
American Society for Cell Biology meeting in Philadelphia, PA, 2014.
9. Zou C, et al. Development and Characterization
of an in vitro Co-culture Angiogenesis Assay System Using hTERT Immortalized
Cells for High Throughput Drug Screening. Poster presented at the annual
American Society for Cell Biology meeting in Philadelphia, PA, 2014.
Even though we don't have to need to many more toxicology asssays on animals. In the process of medical development, when people find some common characteristics of physiological characteristics, pathological and human beings, they will begin the scientific research based on animal experiments.
ReplyDeleteThere is no doubt that this cell lines can promote experiment procedures.--Creative Diagnostics
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