Carcinogenesis vol.27 no.3 pp.374–381, 2006
doi:10.1093/carcin/bgi266
Advance Access publication November 15, 2005
REVIEW
Experimental models of human bladder carcinogenesis
R.A.Crallan, N.T.Georgopoulos and J.Southgate
Jack Birch Unit of Molecular Carcinogenesis, Department of Biology,
University of York, York, YO10 5YW, UK
To whom correspondence should be addressed. Tel: þ44 0 190 432 8705;
Fax: þ44 0 190 432 8704;
Email: [email protected]
Bladder cancer is the fifth most common cancer in the UK,
yet human bladder carcinogenesis remains poorly understood and the response of bladder tumours to radio- and
chemo-therapy is unpredictable. The aims of this article
are to review human bladder carcinogenesis and appraise
the different in vitro and in vivo approaches that have been
developed to study the process. The review considers how
in vitro models based on normal human urothelial (NHU)
cells can be applied to human bladder cancer research. We
conclude that recent advances in NHU cell culture offer
novel approaches for defining urothelial tissue-specific
responses to genotoxic and non-genotoxic carcinogens
and elucidating the role of specific genes involved in the
mechanisms of bladder carcinogenesis and malignant
progression.
Bladder cancer and carcinogenesis
In developed countries, the majority (490%) of bladder cancers are sporadic carcinomas that arise from the urothelium,
the highly-specialized transitional epithelium that lines the
urinary bladder. Urothelial cell carcinoma (UCC or transitional cell carcinoma) is three times more common in men
than in women and is predominantly a disease of the elderly
(http://www.cancerresearchuk.org/aboutcancer/statistics/). As
historically, the molecular pathways leading to the development and progression of muscle-invasive UCC were poorly
understood, UCC has come to represent all urothelial neoplasms, irrespective of their malignant potential. It is now,
however, evident that UCCs arise via two major pathways,
each of which is defined by distinct biological phenotypes and
underpinned by specific genetic lesions. The most common
pathway, which is representative of 70% of UCC at clinical
presentation, leads to the development of low-grade papillary
tumours. Despite their high frequency of recurrence (60–70%)
following cystoscopic removal, these tumours have a relatively low rate of progression (10–20%) to muscle-invasive
disease, although a proportion of tumours may recur at a higher
grade (1). Papillary tumours represent an indolent form of
UCC, yet there remain challenges in predicting the subset of
tumours with malignant potential [e.g. (2)], in order to provide
appropriate disease management. At clinical presentation,
Abbreviations: 4-ABP, 4-aminobiphenyl; HPV, human papillomavirus;
NHU, normal human urothelial; MMC, mitomycin c; MNU, N-methyl-Nnitrosourea; RNAi, RNA interference; UCC, urothelial cell carcinoma.
Carcinogenesis vol.27 no.3 # Oxford University Press 2005; all rights reserved.
a further 20% of patients are found to have muscle-invasive
UCC, which is thought to develop from flat, high grade dysplastic lesions of carcinoma in situ (CIS). This second pathway
represents UCC of high malignant potential, which show high
rates of progression and metastasis, poor response to therapy
and five-year survival rates of only 50%.
A large number of genetic changes have been associated
with the genesis and progression of UCC, many of which
appear to abrogate the G1/S cell cycle checkpoint [reviewed
in (3)]. In particular, loss of p53 function has been linked to the
development of muscle-invasive disease (4) and loss of retinoblastoma protein (pRb) has been associated with metastatic
potential (5). However, no robust, unified model defining the
two pathways has yet emerged. One model receiving current
scrutiny is that activating point mutations in the FGFR3 receptor gene, which are associated with UCCs of low malignant
potential, are mutually exclusive to alterations in the p53 gene.
If verified, changes in FGFR3 and p53 genes, respectively,
could epitomize the two alternative genetic/biological pathways of UCC (5,6).
An important feature of the biology of urothelium that may
influence urothelial carcinogenesis relates to its function as
a self-renewing urinary barrier. Normal urothelium from all
species has an extremely low rate of turnover and virtual
absence of mitoses (8,9), but a high regenerative capacity,
showing rapid proliferation during development and in
response to damage or injury (10). Excreted urinary carcinogens may promote carcinogenesis, not only by direct genotoxic
damage, but also by damaging the epithelium and driving
proliferation towards restoration of a urinary barrier.
A number of risk factors have been strongly linked to the
development of bladder cancer. Tobacco smoking is the highest risk factor, accounting for at least one-third of bladder
cancer cases (11). Typical smokers are two to three times
more likely to develop the disease than non-smokers, and the
risk increases with intensity of smoking (12–15). Tobacco
smoke contains 4000 chemicals, many of which are genotoxins, such as N-nitroso compounds and aromatic amines,
e.g. 4-aminobiphenyl (4-ABP) and o-toluidine. The urine of
smokers has been shown to be mutagenic, containing twice
the total concentration of aromatic amines compared to nonsmokers (16–18). DNA adducted to 4-ABP has been detected
in significantly higher levels in both exfoliated cells in the
urine and urothelium or UCC biopsies from smokers compared
with non-smokers (19–23) [reviewed in (24)]. 4-ABPhaemaglobin adducts are also elevated in the blood of smokers;
these adducts are potential biomarkers for estimating individual exposure to bladder carcinogens (25,26) [reviewed
in (27)]. Mutation hotspots in the p53 gene of human bladder
tumours correlate with the DNA-binding spectrum of 4-ABP,
providing further evidence for the involvement of this important tobacco carcinogen in urothelial carcinogenesis (28).
Smoking appears to influence p53 mutations and is associated with invasive, high grade UCCs; by contrast, FGFR3
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Experimental models of human bladder carcinogenesis
mutations are not associated with smoking status and may
result from endogenous DNA damage (29). Non-genotoxins
in tobacco smoke may also contribute to bladder carcinogenesis by promoting tumour growth, for example by increasing
cell proliferation or suppressing the immune response (30).
Domestic or occupational use of hair dyes has been suggested as a risk factor, particularly in women, who have a poorer
survival rate than men (31). Exposure to industrial chemicals
[reviewed in (32)] and drugs such as cyclophosphamide (33)
and phenacetin (34) also increase bladder cancer risk. Chronic
irritation or infection of the bladder has been implicated,
possibly due to the production of nitroso compounds or reactive oxygen species in the bladder, combined with increased
proliferation in response to injury (35–37). In areas where
infestation with the Schistosomiasis haematobium parasite
is endemic, such as Egypt, infection is associated with an
increased risk of developing squamous cell carcinoma (SCC),
a histopathologically distinct form of bladder cancer (38)
[reviewed in (39)]. The development of SCC may be related
to dietary factors, e.g. Vitamin A levels, which can affect
differentiation of the urothelium (40,41). Genetic factors,
such as polymorphisms in genes encoding carcinogen metabolizing enzymes or DNA repair proteins, may contribute to
inter-individual variation in bladder cancer susceptibility (42).
Paradoxically, genotoxins are also used to treat bladder cancer. Intravesical cytotoxic drugs, such as mitomycin C
(MMC), a crosslinking antibiotic (43), are instilled into the
bladder following transurethral resection of poorly differentiated, early stage tumours in order to reduce the risk of recurrence (44). Invasive bladder tumours may be treated with
radiotherapy. However, the response of bladder tumours to
both chemotherapy and radiotherapy is highly unpredictable.
Resistance to chemotherapy may arise from over-expression
of genes, such as the multidrug resistance gene mdr1 (45,46).
The p53 status of tumour cells is also implicated in therapy
response, but its effect is unclear. p53-null UCC tumours
have been reported to show increased sensitivity to some
chemotherapeutic agents, but not others (47–49). Increased
survival following radiotherapy of bladder tumours with
altered p53 has been reported (50), whereas other studies
have demonstrated the opposite effect (51); yet others have
found no relationship between p53 status and response to
radiochemotherapy (52).
Animal models of bladder cancer
Animal models of cancer have been important for demonstrating that carcinogenesis is a multistep process comprising three
key stages: initiation, the irreversible fixation of a genetic
alteration; promotion, the reversible expansion of the tumour
cell population; and progression, the irreversible transition to a
malignant, invasive tumour (53–55).
In many models of bladder carcinogenesis, multiple doses
of carcinogens have been instilled directly into the bladders
of rodents and dogs. In 1972, Hicks and Wakefield used
four doses of N-methyl-N-nitrosourea (MNU) to rapidly
induce bladder cancer in rats, demonstrating that it is a complete carcinogen. However, the doses used were very high
(100 mM) and histological analysis of this dose in rat
bladders revealed areas of necrosis, desquamation and haemorrhage followed by urothelial hyperplasia (56,57). This suggests that the compound induced cytotoxic damage to the
urothelium, which would have invoked a ‘wound response’,
driving rapid proliferation and allowing the accumulation and
perpetuation of mutations. In the human bladder, it is likely
that the urothelium is chronically exposed to sub-cytotoxic
doses of genotoxins and, hence, acute exposure may not be
representative of the process in man. Nevertheless, MNU has
been used as the initiating agent in many rodent studies in
order to test compounds for their ability to promote bladder
tumorigenesis (58–61). Rodent models have also been used
to study the contribution of DNA adduct burden to tumour
incidence (62).
However, rodent models have produced false positive results. For instance, the artificial sweetener, sodium saccharin,
was shown to induce bladder tumours in several studies
(58,63,64). The mechanism was later shown to be due to the
formation of calculi in the urinary tracts of rats, resulting in
physical damage to the urothelium and a proliferative wound
response that led to the promotion of coincidental or dormant
mutations (65). Saccharin exposure does not lead to calculi
formation in the human bladder due to differences in renal
physiology, although the carcinogenic potential of the compound is still under debate. Thus, differences in physiology,
metabolism and urinary pH between rodents and humans,
together with the lower threshold for immortalization in
rodent models due to constitutive telomerase activity in all
somatic cells, strongly suggest that rodent models of bladder
cancer are not ideal for studying mechanisms of human
carcinogenesis (30,66).
In order to overcome the species differences inherent in
rodent models, bladder carcinogenesis has also been studied
in dogs. Owing to similarities with human metabolic processes, dogs are more suitable for experiments with carcinogens that require bioactivation. 4-ABP and its metabolites have
been administered orally, intravenously and transurethrally to
beagle dogs in order to study urothelial adduct formation,
repair and tumour incidence (67–69). Although these studies
have been useful in elucidating the mechanisms of bladder
carcinogenesis mediated by 4-ABP, experiments on dogs
incur both financial and ethical constraints.
The most useful animal model to emerge in recent years is
the transgenic mouse, in particular the development of mice in
which the transgene is driven from the tissue-specific uroplakin II gene promoter, so that expression is restricted to the
urothelium (55,70–73). Mice expressing constitutively activated H-Ras developed urothelial hyperplasia and low-grade
superficial papillary tumours (55), whereas mice expressing
the SV40 T antigen, which functionally inactivates both p53
and pRb, developed CIS and invasive bladder tumours (70).
These models afford insight into the molecular basis of
urothelial cell tumorigenesis and the pathways of bladder
cancer progression in man and provide direct experimental
evidence that specific genetic lesions give rise to phenotypically distinct bladder tumours.
Bladder cancer cell lines
A number of established cell lines have been developed from
rodent urothelial cells, facilitated by tissue accessibility and
the propensity of rodent cells to undergo spontaneous transformation more readily than human cells (74). Rat urothelial
cells have been used to show that agents such as MNU and
hydrogen peroxide (H2O2) can cause malignant transformation
of normal, non-tumorigenic and of low-grade tumour cells
(75–77). However, and in light of the unsuitability of the rat
375
R.A.Crallan et al.
Normal
Urothelium
Molecular Phenotype
Normal Urothelial Cells
Induction of
Differentiation
Isolation
& Culture
Genetic Manipulation
(“Para-malignant” cells)
Immortalisation
(HPV E6
hTERT)
Urothelial Cell
Carcinoma (UCC)
Isolation
& Culture
Comparison in situ
Defined genetic
alterations
3D
(DN mutants
Reconstruction
siRNA)
(Organotypic
Culture)
Established
UCC lines
(RT4, RT112, EJ)
Comparison in vitro
Structure/Function
relationships
Normal
Behaviour
(NHU)
Malignant
Behaviour
(EJ)
Reconstruction
Fig. 1. The NHU Cell Culture System. Schematic diagram summarizing the use of urothelial cell cultures for understanding urothelial carcinogenesis. Normal
and malignant urothelial behaviour can be studied in situ (blue), for instance by immunohistochemical studies. Upon isolation and culture, NHU cells can be
maintained as highly proliferative, finite cell lines. By means of genetic manipulation (viral transduction and induction of defined genetic alterations),
’paramalignant’ cells can be established (yellow)—see text for detailed discussion. Studying these cells and comparing them to their well-characterized,
malignant counterparts (RT4, RT112, EJ) has proven to be very useful in understanding the role of early genetic events in malignant transformation.
The ability to induce full maturation/differentiation of urothelial cells and 3D reconstitution of normal and tumour cells in organ culture (green) both
attests to the validity and strength of the urothelial system for such studies and renders the urothelial system unique among other human tissue
culture models.
model for understanding human carcinogenesis discussed
above, the relevance of the rodent cell lines to normal and
malignant human urothelial cells is questionable.
Many established cell lines have been derived from human
UCC, reflecting access to tumour resection specimens and the
immortalized nature of tumour-derived cells. Cell lines representing tumours of different grades and stage have been
developed and variably characterized [see (78) for review of
22 cell lines] and have been shown to retain characteristics of
the originating tumours. This is particularly exemplified in
orthotopic nude mice models (79,80) and human in vitro
organotypic systems (81,82), where UCC-derived cell lines
have been shown to develop tumours that recapitulate the
differentiation (grade) and invasion (stage) characteristics of
the originating tumour. However, the success rates for generating cell lines from primary tumour material are very low and
therefore the lines represent a very select subset of tumours
with the potential to survive and adapt to a cell culture environment. Of all the human UCC-derived lines, only the RT4
cell line represents a well-differentiated papillary non-invasive
(G1 pTa) tumour, which as discussed above is the most
common form of human bladder cancer.
376
Bladder cancer cell lines have been invaluable for studying
bladder tumour cell behaviour. For example, analysis of a
panel of invasive UCC-derived cell lines helped define the
functional significance and inter-relationships of pRb and
p53 pathways in UCC development, and provided important
insight into how the biology of UCC is governed by the
mechanism of pathway inactivation (83). Nevertheless, UCCderived cells are abnormal, with complex karyotypes reflecting accumulated genetic aberrations and although uncloned
cell lines potentially represent the genetic and phenotypic
biodiversity of the tumour population, they may exhibit genetic/phenotypic drift due to selection pressures imposed during
extensive cell culture. Thus, data arising from studies using
cancer cell lines may be idiosyncratic and in certain cases
conflicting. For instance, p53-defective human bladder cancer
cell lines have been reported as radiosensitive in one study
(84), radioresistant in another (85), whereas a third study
reported no correlation between p53 status and sensitivity
to methotrexate, adriamycin or cisplatin in nine bladder UCC
cell lines (86). It is also worth highlighting the importance of
pedigree, as cross-contamination between cell lines (of urothelial or non-urothelial derivation) can lead to confounding
Experimental models of human bladder carcinogenesis
results and the use of cell lines with characterized marker
expression or sourced from authenticated cell banks (e.g.
European Collection of Cell Cultures at http://www.ecacc.
org.uk/ or American Type Culture Collection at http://www.
lgcpromochem.com/atcc/) should be mandatory [discussed
in (87–89)].
Normal human urothelial cells
Although urothelium has a low constitutive turnover rate,
during urothelial repair, cells from all layers of the urothelium
are recruited into the cell cycle (90). This regenerative ‘wound
response’ is exploited in the isolation and growth of normal
human urothelial (NHU) cells in vitro.
Traditionally, epithelial cells have been refractory to growth
in monolayer cell culture, due to their propensity to become
overgrown by competing stromal cell populations. A breakthrough was the development of low calcium, serum-free
growth media to promote growth of normal human keratinocytes, at the expense of serum-dependent fibroblast growth.
This led to the development of techniques to isolate and culture
NHU cells from surgical specimens of bladder, ureter and
renal pelvis (91–97).
NHU cells, cultured as monolayers in low calcium
(0.09 mM) keratinocyte serum-free medium containing bovine
pituitary extract, recombinant epidermal growth factor and
supplemented with cholera toxin, have been characterized
extensively (96,97). NHU cell cultures display a rapid (15 h)
proliferation rate during log phase growth and are contactinhibited at confluence, but may be propagated as finite cell
lines through at least 20 population doublings by serial passage. The removal of exogenous growth factors from the
medium has been shown to make little difference to the exponential growth rate of NHU cell cultures, demonstrating that
NHU cells stimulate their own proliferation in an autocrine
or juxtacrine fashion (98), which is mediated through the
EGF receptor (98–102). NHU cell cultures exhibit a basal/
intermediate urothelial cell phenotype, but both stratification
(96) and differentiation (103–105) may be induced, attesting to
the normal differentiation potential of NHU cell cultures.
Another feature of normal cells is that cultures derived from
different donors retain the genotype of the tissue donor, so
inter-individual variation can be studied. For example, DNA
repair gene expression was shown to vary between six primary
urothelial cell lines cultured under identical conditions; such
differences may reflect differential susceptibility to bladder
cancer (106). This inter-individual variation between cell
lines also implies that several tissue donors should be used
when assessing genotoxic response.
NHU cell lines have been used to investigate two important
aspects of bladder cancer. First, studying the effects of carcinogens has elucidated possible mechanisms of bladder cancer
development. Bladder carcinogens investigated include
4-ABP, the carcinogen implicated in tobacco-induced bladder
cancer, MNU, a direct acting genotoxin used to develop rodent
models of bladder cancer and H2O2, implicated in the development of bladder cancer due to infection. Typically, such
studies have investigated the effects of genotoxic agents on
cell morphology, proliferation, apoptosis and cell cycle arrest.
For instance, Reznikoff et al. (107) showed that the response of
NHU cells to MNU involved initial growth delay and subsequent recovery of morphologically normal cells, and Chien
et al. (108) reported p53-independent G2 check-point arrest
and induction of senescence after treatment with H2O2.
Second, studying the effects of therapeutic agents such as
g-radiation and MMC on normal cells may help develop treatment strategies for bladder cancer. For example, MMC has
been reported to induce cell cycle arrest with stabilization
of p53 and upregulation of p21 proteins (109), whereas
g-radiation has been reported to induce apoptosis, premalignant changes and the expression of c-myc and bcl-2,
with inter-individual variation in responses (110–112).
Genetically manipulated human urothelial cells
A disadvantage of using NHU cells is that they can only be
subcultured a finite number of times before undergoing spontaneous replicative senescence (95). The role of telomere
shortening in this process is unclear and it is thought that
replicative senescence is due to cellular accumulation of the
cyclin-dependent kinase inhibitor protein, p16INK4 (113,114).
An understanding of the mechanisms leading to accumulation
of p16INK4 protein in cultured cells is incomplete, although it
may at least in part represent a culture artefact induced by
stress, such as DNA damage or sub-optimal culture conditions
(115). Nevertheless, it is likely that bladder tumorigenesis
in vivo requires that this senescence block is overcome (116).
To establish long-term human urothelial cell lines, oncogenic viruses, such as SV40 and high-risk human papillomavirus (HPV), or their oncogenic products, large T antigen
and E6/E7 oncoproteins, respectively, have been exploited as
immortalizing agents (117–119). The derived cell lines have
been used to investigate the role of genotoxins, including
4-ABP, in multistage carcinogenesis (120–123) and the effects
of p53 loss on response of cells to chemo- and/or radiotherapies. Loss of functional p53 has been correlated with
resistance to both radiation and methotrexate in HPVimmortalized urothelial cells in vitro (124,125). By contrast,
Diggle et al. (109) reported that HPV16 E6 expression sensitized urothelial cells in vitro to treatment with MMC or
g-radiation.
Although these modified cell lines retain urothelial characteristics, it is clear that major signalling pathways controlling
decision-making in the context of cell cycle progression, proliferation and/or apoptosis have been inactivated. Recent studies have demonstrated that HPV16 E6-transformed NHU cells
exhibit increased susceptibility to growth inhibition and apoptosis signals such as those triggered by members of the Tumour
Necrosis Factor (TNF) superfamily of proteins (126,127), the
classical inducers of apoptotic cell death in both immunocytes
and epithelial cells. Thus, in addition to offering the advantage
of extended life-span, these immortalized cells have been useful in understanding how early events in malignant transformation may influence lympho-epithelial interactions in the
context of tumour surveillance and how genetic changes affect
urothelial cell responses to signals associated with proliferation and/or cell death.
Despite these advantages, however, the immortalization
strategy has two inherent limitations. First, although the
derived cells are non-tumorigenic, they are, by definition, no
longer ‘normal’, as the oncogenic viral proteins disable both
the critical p53 and pRb tumour suppressor pathways. For
instance, loss of p53 function favours genetic instability and
allows immortalized cells to accumulate chromosomal abnormalities, leading to the development of dominant, karyotypically abnormal transformed sub-populations (109,128–130).
377
R.A.Crallan et al.
Second, these cells have no ‘defined’ genetic changes and a
number of signaling pathways have been inactivated simultaneously. For example, in addition to causing the degradation of
p53 via ubiquitination, the HPV 16 E6 oncoprotein sensitizes
cells to apoptosis (131) and activates the catalytic subunit of
telomerase (hTERT) (132). Therefore, in order to understand
how specific genetic changes influence urothelial cell homeostasis and to provide a better-defined in vitro model of urothelial carcinogenesis, more subtle genetic manipulations are
required. A number of approaches for such studies have been
developed, including the use of dominant-negative mutant
proteins to disable specific tumour suppressor function.
NHU cells are susceptible to transduction by amphotropic
retroviruses, which provides a highly efficient (490%) method
to introduce gene sequences of interest into NHU cells. This
permits, for example, the effects of disabling key tumour
suppressor genes, such as p53 and p16, to be studied. We
have used this approach to transduce NHU cells and select
stable ‘paramalignant’ human urothelial sublines expressing
either a dominant-negative p53 miniprotein (HU-p53DD) or a
p16-insensitive CDK4 mutant (HU-CDK4mut), respectively
(127). The functionality of the sublines was demonstrated by
the inability of HU-p53DD cells to transactivate p21 and
hyperphosphorylation of retinoblastoma protein in HU-CDK4
cells (127). The use of retroviruses designed with different
antibiotic selection cassettes offers the potential to look at
combinations and sequences of genetic events, such as the
loss of p53 from a p16-disabled background. In our hands,
however, cells could not survive the simultaneous loss of p53
and p16 functions, suggesting that other changes, such as
telomerase activation, may be required (127). We have also
demonstrated that loss of p16, but not p53, function altered
the susceptibility of NHU cells to apoptosis induced by CD40,
a member of the TNF receptor family (127). Although the
importance of p53 and p16 loss in bladder carcinogenesis is
well established (133), such studies may provide an understanding of the underlying molecular mechanisms of progression to malignancy.
The establishment and characterization of ‘paramalignant’
human urothelial cells with engineered genetic alterations
(Table I) is a promising tool to understanding urothelial carcinogenesis. This approach will be revolutionized by the use of
siRNA technologies to develop cells with ‘knocked-down’
candidate tumour suppressor gene activity in a NHU cell
background. Such strategies will contribute significantly to
our understanding of the influence of specific genetic changes
in urothelial tumorigenesis and should have application in
identifying novel, relevant chemo- and immuno-therapeutic
targets.
Collectively, functional protein inhibition and phenotypic
modification studies may provide a means to elucidate the
multistep pathways leading from normal cells to cancer cells
and identify potential therapeutic targets. As the genotypic
and, consequently, phenotypic heterogeneity of tumours dictates that effective remedies may require patient-specific
application, such in vitro models could prove invaluable for
the development of more effective treatments on a tumourspecific basis.
In conclusion, the engineering of ‘designer’ human urothelial
cells has an, as yet, largely unexplored potential to improved
specificity and physiological relevance over the experimental
models of yesterday, although the clinical relevance of findings derived from these models will need to be determined by
their validation in an in vivo setting.
New developments and future prospects
Acknowledgements
Investigation of protein function by inhibition of activity has
been critical to understanding a variety of biological processes,
with antisense oligonucleotides, dominant-negative mutants
and knockout transgenic animals being some of the most frequently employed methodologies. More recently, a new strategy has emerged, namely post-transcriptional gene silencing
or RNA interference (RNAi), using small interfering RNA
(siRNA) molecules (134). Combination of this powerful
technology with the use of viral vectors for efficient delivery
and stable siRNA expression [for an example see (135)] has
opened new avenues for investigation, both in the field of
functional genomics (136) and in clinical therapeutics. RNAi
is characterized both by choice of targets and a high degree of
specificity, and has been used successfully to study the consequences of inactivating a wide range of cancer-associated
genes and signalling proteins, including Ras, Bcl-2 family
members, cell cycle regulators, vascular endothelial growth
factor and viral oncogenes (137). Importantly, there are a
number of examples where RNAi has shown efficacy at reducing the tumour burden in experimental cancer models in vivo
(137), providing great promise for future RNAi-based cancer
therapies.
R.A.C. was supported by a Biotechnology and Biological Sciences Research
Council (BBSRC) Industrial CASE Studentship with GlaxoSmithKline and
N.T.G. is a postdoctoral Research Fellow funded by the Engineering and
Physical Sciences Research Council (EPSRC), Grant GR/S62338/01. J.S. is a
Research Professor supported by York Against Cancer.
378
Table I. Cell lines derived from genetically-manipulated human
urothelial cells
Cell line
Protein expressed/modification
Reference(s)
SV-HUC
UROtsa
Simian virus 40 T-antigen
Temperature-sensitive simian
virus 40 large T-antigen
HPV type 16 E6 protein
HPV type 16 E7 protein
Dominant-negative p53 mutant
p16-insensitive CDK4 mutant
(111)
(112)
E6-HUC & HU-E6
E7-HUC
HU-p53DD
HU-CDK4mut
(104,113,120)
(113)
(121)
(121)
Conflict of Interest Statement: None declared.
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Received August 11, 2005; revised October 31, 2005;
accepted November 2, 2005
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