Download Urothelial Cells Malignantly Transformed by

TOXICOLOGICAL SCIENCES 94(2), 293–301 (2006)
doi:10.1093/toxsci/kfl108
Advance Access publication September 15, 2006
Urothelial Cells Malignantly Transformed by Exposure to
Cadmium (Cd+2) and Arsenite (As+3) Have Increased
Resistance to Cd+2 and As+3-Induced Cell Death
Seema Somji,1 Xu Dong Zhou, Scott H. Garrett, Mary Ann Sens, and Donald A. Sens
Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202
Received August 2, 2006; accepted September 11, 2006
This laboratory has shown that both Cd+2 and As+3 can
malignantly transform human urothelial cells. The present study
examined metal resistance and the mechanism of cell death when
the parental and malignantly transformed UROtsa cells were
exposed to Cd+2 and As+3. It was shown that the malignantly
transformed UROtsa cells were more resistant to the toxic effects
of both metals. The assessment of the mode of cell death demonstrated that the parental UROtsa cells died by both apoptosis and necrosis when exposed to either metal. It was shown
that apoptosis was the more prominent mechanism of cell death,
accounting for over 50% of cell death. Apoptotic cell death
was determined by the observation of fragmented nuclei using
4#,6-diamidino-2-phenylindole staining, the formation of a DNA
ladder, and the detection of cleaved caspase-3 and caspase-9
products in the cell lysates. Necrotic cell death was determined by
measuring the release of lactate dehydrogenase into the growth
medium. It was determined that the extent of apoptosis of the
malignantly transformed UROtsa cells was decreased and that the
extent of necrosis was increased compared to the parental UROtsa
cells. These observations are consistent with in vivo studies which
suggest that As+3 can act as a tumor promoter during the
regeneration of the bladder urothelium. The present in vitro
studies suggest that As+3-induced cytotoxicity could set the stage
for tissue repair due to its own inherent toxicity to normal
urothelium, and then subsequently act as a tumor promoter
during the regeneration process through the stimulation of the
regrowth of cells that have gained increased resistance to As+3.
Key Words: arsenite; cadmium; urothelium; bladder cancer;
apoptosis; necrosis.
Transitional cell carcinoma of the bladder is the fourth most
common cancer in men and the fifth in women in Western
countries (Johansson and Cohen, 1997). Historically, bladder
cancer is the first cancer in which industrial carcinogens were
found to play the major role in disease causation. This was first
1
To whom correspondence should be addressed at Department of Pathology,
School of Medicine and Health Sciences, University of North Dakota, 501 N.
Columbia Road, Grand Forks, ND 58202. Fax: (701) 777-3108. E-mail:
[email protected].
observed by Rehn in 1895 when a link was demonstrated
between exposure to aromatic amines and development of
bladder cancer in factory workers (Rehn, 1895). This association
was subsequently confirmed in both animal models and humans
working in industries that involve exposure to aromatic amines
(Case et al., 1954; Hueper et al., 1938). In more recent times, an
association between cigarette smoking and bladder cancer was
found, with some reports suggesting a two- to fourfold increased
risk and that 50% of the bladder cancers in men would not occur
in the absence of cigarette smoking (Clavel et al., 1989; Morrison
et al., 1984). Most recently, epidemiologic evidence has provided substantial evidence linking the ingestion of arsenic in
drinking water with the development of bladder cancer
(Steinmaus et al., 2000). Epidemiologic studies have shown a
strong association between arsenic ingestion from contaminated
drinking water and the development of bladder cancer in Taiwan
(Chiou et al., 1995), Argentina (Hopenhayn-Rich et al., 1996),
Chile (Smith et al., 1998), and Japan (Tsuda et al., 1995). Arsenic
is classified by the International Agency for Research on Cancer
as a human carcinogen (IARC, 1980), and it is presently first in
priority among a listing of the top 20 hazardous substances by the
Agency for Toxic Substances and Disease Registry and the U.S.
Environmental Protection Agency (ATSDR, 1997). Another
heavy metal, cadmium (Cdþ2), is also a known human carcinogen that has been associated with the development of bladder
cancer (Siemiatycki et al., 1994; Waalkes, 2000), although the
epidemiologic data is much less extensive. Like arsenic,
cadmium has been classified by the International Agency for
Research on Cancer as a human carcinogen (IARC, 1993).
There are limited human models of bladder cancer available
for study where malignant transformation has been induced by
environmental agents. This laboratory has recently directly
malignantly transformed an immortalized cell culture of human
urothelial cells (UROtsa) with both Cdþ2 and Asþ3 (Sens et al.,
2004). This required extended exposure of the cell line to 1lM
Cdþ2 or Asþ3. The resulting transformed cells were shown to
have increased growth rates and to readily form colonies in soft
agar when compared to untreated control cells. The transformed
cells arising from treatment with both Cdþ2 and Asþ3 were
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shown to form tumors when injected sc into immunocompromised (nude) mice. The histology of the tumor heterotransplants
displayed the features expected of human transitional cell
carcinoma of the bladder. An interesting observation in this
study was that before the resulting cell cultures gained the ability
to form colonies in soft agar, and subsequently tumors in nude
mice, both cell lines experienced two similar episodes of Asþ3and Cdþ2-induced cell death and subsequent regrowth of the
culture to confluency. This may also occur within the human
bladder under conditions of metal exposure where the urothelium
undergoes cell death followed by proliferation to a complete
transitional epithelium. The role of cell proliferation preceded by
toxicant-induced cell death has been intimately associated with
the models of carcinogenesis particularly in the liver (Oliver and
Roberts, 2002) and the cancer-causing effect of one arsenic
compound, dimethylarsenic acid (DMA) may be related to the
observed cycle of cell death and regeneration (Cohen et al.,
2001). The observed cycle of proliferation preceded by metalinduced cell death during the UROtsa in vitro transformation
process raised several questions. First, does the transformation
process alter the susceptibility of the transformed cells to further
exposure to Cdþ2 and Asþ3, thereby enhancing the accumulation
of transformed cells within the urothelium? Second, what is the
mechanism of cell death in human urothelial cells exposed to
Asþ3 and Cdþ2, does the transformation process alter the
mechanism of cell death in human urothelial cells and is it
the same for both metals? Lastly, does the intracellular level of
the metal-binding protein, metallothionein (MT), influence the
malignant transformation of urothelial cells exposed to Cdþ2 or
Asþ3? The present study addresses these questions.
MATERIALS AND METHODS
Cell culture. Stock cultures of the parent UROtsa cells and UROtsa cell
lines malignantly transformed with either 1lM Cdþ2 or Asþ3 were maintained
in 75 cm2 tissue culture flasks in Dulbecco’s modified Eagle’s medium
containing 5% vol/vol fetal calf serum as described previously (Rossi et al.,
2001; Sens et al., 2004). Cultures were incubated at 37°C in a 5% CO2: 95%
air atmosphere. The cells were fed fresh growth medium every 3 days, and
when confluent, the parental UROtsa cells were subcultured at a 1:4 ratio
and the transformed cells at a 1:20 ratio using trypsin-EDTA. The Cdþ2and Asþ3-transformed cell lines were serially passaged 10 times in Cdþ2- and
Asþ3-free growth medium before use in any experimental protocols.
The parental cell line is defined by the designation UROtsa, cells
transformed with Cdþ2 on serum-containing growth medium by the designation
URO-CDSC, and cells transformed with Asþ3 on serum-containing growth
medium by the designation URO-ASSC.
Visualization of DAPI-stained cells. The effect of metal treatment on cell
viability and the number of fragmented (apoptotic) nuclei were visualized
microscopically using 4#,6-diamidino-2-phenylindole (DAPI)-stained nuclei as
described previously by this laboratory (Somji et al., 2004). At the indicated
time points, wells containing the monolayers were rinsed with phosphatebuffered saline (PBS), fixed for 15 min in 70% ethanol, rehydrated with 1 ml
PBS, and stained with 10 ll DAPI (10 lg/ml in distilled water).
MTT assay for cell viability. Cell viability, as a measure of cytotoxicity, was
determined by measuring the capacity of the cells to reduce MTT (3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to formazan (Rossi et al.,
2002). Triplicate cultures were analyzed for each time point and concentration.
DNA laddering and Lactate Dehydrogenase release. DNA laddering was
used to confirm the correlation of apoptosis with the presence of fragmented
nuclei observed using DAPI staining (Somji et al., 2004). Briefly, at each time
point, adherent and detached cells were collected and combined from each
well, centrifuged, and the pellet resuspended in lysis buffer. The cell lysate was
centrifuged and the supernatant was incubated with 200 lg/ml proteinase K for
1 h at 50°C. The DNA was extracted with phenol:chloroform:isoamyl alcohol
(25:24:1 vol/vol/vol) and precipitated overnight with absolute ethanol in the
presence of 20 lg glycogen. The DNA pellet was washed twice with 70%
alcohol, air dried, and dissolved in Tris-EDTA buffer. After treatment with
ribonuclease A for 1 h at 50°C, the DNA was loaded onto a 2% (wt/vol) agarose
gel containing ethidium bromide.
Lactate dehydrogenase (LDH) release was used to confirm the correlation of an
absence of fragmented nuclei on DAPI-stained cells with a necrotic mechanism of
cell death. The release of LDH from cells was determined by the Cyto Tox 96 assay
kit (Promega, Madison, WI) as described previously (Somji et al., 2004). Briefly,
50 ll of the cell culture supernatant was transferred to a 96-well enzymatic assay
plate. Reconstituted substrate mix (50 ll) was added to each sample and the
enzymatic reaction was allowed to proceed for 30 min at room temperature in the
dark. The assay was stopped by adding 50 ll of the stop solution (1M acetic acid),
and the plate was read at 490 nm using an ELISA plate reader.
Determination of caspase-3 and caspase-9 activation. Cells were rinsed
with phosphate buffered saline and harvested in 3-[(3-cholamidopropul)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) cell extract buffer (50mM
homopiperazine-1, 4 bis(2-ethanesulfonic acid)/NaOH, pH 6.5, 2mM EDTA,
0.1% CHAPS, 5mM DTT, 20 lg/ml Leupeptin, 10 lg/ml pepstatin, 10 lg/ml
aprotinin, and 1mM PMSF) (Cell Signaling Technology, Beverly, MA). The
harvested cells were freeze-thawed three times followed by centrifugation at
14,000 rpm. The pelleted debris was discarded, and the supernatant was used for
analysis. The concentration of protein in the samples was determined by the BCA
protein assay (Pierce Chemical Co., Rockford, IL). Ten micrograms of total
cellular protein was separated on 12% SDS-containing polyacrylamide gel and
electrophoretically transferred to a hybond-P polyvinylidine difluoride membrane (Amersham Biosciences, Piscataway, NJ). Membranes were blocked in
Tris buffered saline containing 0.1% Tween-20 (TBS-T) and 5% (wt/vol)
nonfat dry milk for 1 h at room temperature. After blocking, the membranes
were probed with the appropriate primary antibody (Caspase-3 and Caspase-9,
Cell Signaling Technology; Actin, Stressgen, Ann Arbor, MI) overnight at 4°C
in antibody dilution buffer (TBS-T) containing 5% nonfat dry milk. Following
three washes, the membrane was incubated with the secondary antibody for 1 h
at room temperature. The blots were visualized using the Phototope-HRP
Western blot detection system (Cell Signaling Technology).
MT protein determination. The immuno-blot protocol used for the
determination of the coexpressed levels of the MT-1 and MT-2 protein in cell
lysates has been described previously by this laboratory (Garrett et al., 1998).
The MT-1/2 proteins were detected by immunoblotting using a mouse antihorse
antibody (DAKO-MT, E9, Dako, Carpinteria, CA) as the primary antibody.
Statistical analysis. All cell culture experiments were performed in
triplicate and the results are expressed as the standard error of the mean.
Statistical analyzes were performed using Systat software using separate
variance t-tests, ANOVA with Tukey post hoc testing. Unless otherwise stated,
the level of significance was 0.05.
RESULTS
Visibility of Parent and Transformed UROtsa Cells
Exposed to Cadmium and Arsenite
The resistance of cadmium and arsenite-transformed cells
to increasing concentration of each metal was assessed by
þ3
As
AND Cd
þ2
-INDUCED UROTHELIAL CELL DEATH
295
measuring cell viability. For this purpose, confluent cultures of
parent and transformed UROtsa cells were exposed to increasing concentrations of Cdþ2 or Asþ3 for 24–48 h and cell
viability was determined using the MTT assay. The results of
this determination showed that Cdþ2-transformed cells were
more resistant to the cytotoxic effects of Cdþ2 compared to the
parental cells (Figs. 2A–2C). Exposure of the URO-CDSC cells
to the highest level of Cdþ2 utilized in the study (27lM) for
48 h resulted in only a 50% loss in cell viability compared to
the exposed parental cells. The URO-ASSC cells were modestly more resistant to Asþ3-induced cell death than the parental UROtsa cells (Figs. 2D–2F). However, by 36 h, both
the parental as well as the Asþ3-tranformed cells were sensitive
to the cytotoxic effects of Asþ3.
Apoptosis and Necrosis of UROtsa Cells
Exposed to Cadmium
One of the goals of this study was to determine if Cdþ2
elicited apoptosis in UROtsa cells. The concentration range and
time course of cadmium exposure for the determination of
cadmium-induced apoptosis and necrosis was determined from
the viability assays. This is important since the sensitivity of
the assays (DNA laddering and caspase activation) used to
confirm apoptosis require a significant number of apoptotic
cells. The initial step was to determine if apoptosis did occur in
parental UROtsa cells exposed to Cdþ2. Evidence that
apoptosis was active in Cdþ2-exposed UROtsa cells was
obtained by observing triplicate cultures of confluent UROtsa
cells exposed to various concentrations of Cdþ2 over time and
staining the cells with DAPI when a majority of the cell
population acquired a ‘‘rounded’’ morphology. The fluorescent
staining of nuclei using DAPI has been used previously by this
laboratory to microscopically visualize the fragmented and
condensed nuclei that are characteristic of apoptotic cells
(Somji et al., 2004). This determination showed that 17.5 ±
1.4% of the UROtsa cells had condensed and fragmented nuclei
when they were exposed to 16lM Cdþ2 for 36 h (Fig. 1A). It
was shown that exposure of parental UROtsa cells to 27lM
Cdþ2 resulted in the formation of a DNA ladder (Fig. 3A) and
cleavage of caspases 3 and 9 (Fig. 4A). An analysis of LDH
release into the growth medium showed a maximum release of
40% of total LDH from the cells under conditions of Cdþ2
exposure that resulted in a total loss of cell viability (Fig. 5A).
Apoptosis and Necrosis of UROtsa Cells Exposed to Arsenite
Using an identical approach to that described above for
cadmium, it was first determined if Asþ3 elicited apoptosis in
UROtsa cells and then a time course was defined for the
determination of arsenite-induced apoptosis and necrosis. It
was shown through DAPI staining that 14.6 ± 1.2% of UROtsa
cells exhibited frequent profiles of condensed and fragmented
nuclei when exposed to 8lM Asþ3 for 24 h, features consistent
with cells undergoing apoptosis (Fig. 1B). It was shown that
FIG. 1. DAPI staining of UROtsa parent and transformed cells. UROtsa
parent and transformed cells were exposed to Cdþ2 or Asþ3 and visualized by
light microscopy. At the first sign of cell toxicity (rounding of cells), the cells
were fixed and stained with the nuclear dye, DAPI. Nuclear morphology was
visualized by fluorescent microscopy. Arrows indicate fragmented nuclei. (A)
UROtsa parent cells treated with 16lM Cdþ2 for 36 h. (B) Urotsa parent
cells treated with 8lM Asþ3 for 24 h. (C) Cadmium-transformed UROtsa cells
treated with 20lM Cdþ2 for 36 h. (D) Arsenite-transformed UROtsa cells
treated with 12lM Asþ3 for 36 h.
exposure of parental UROtsa cells to 16lM Asþ3 resulted in
the formation of a DNA ladder (Fig. 3B) and cleavage of
caspases 3 and 9 (Fig. 4B). An analysis of LDH release into the
growth medium showed a maximum release of 30% of total
LDH from the cells under conditions of Asþ3 exposure that
resulted in a total loss of cell viability (Fig. 5B).
Apoptosis and Necrosis of Cd þ2-Transformed UROtsa
(URO-CDSC) Cells Exposed to Cd þ2
Another goal of this study was to determine if UROtsa cells
malignantly transformed through exposure to Cdþ2 would have
an alteration in the mechanism of Cdþ2-induced cell death
compared to that of the parental cells exposed to Cdþ2. It was
first determined using DAPI staining as described above to
determine if putative apoptotic cell profiles could be found in
URO-CDSC cells exposed to Cdþ2. It was shown that 12 ±
1.2% of URO-CDSC cells exhibited profiles of condensed and
fragmented nuclei when exposed to 20lM Cdþ2 for 36 h,
features consistent with cells undergoing apoptosis (Fig. 1C).
DNA laddering and caspase activation could be shown for the
URO-CDSC cells exposed to 27lM Cdþ2 (Figs. 3C and 4C),
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FIG. 2. Effects of Cdþ2 or Asþ3 on the viability of UROtsa parent and transformed cells. Confluent cultures of parent UROtsa cells and Cdþ2-transformed UROtsa cells (URO-CDSC) were exposed to various concentrations of Cdþ2 for 24 (A), 36 (B), and 48 (C) h. Confluent cultures of parent UROtsa- and
Asþ3-transformed UROtsa (URO-ASSC) cells were exposed to various concentrations of Asþ3 for 24 (D), 36 (E), and 48 (F) h. Cell viability was determined by measuring the capacity of the cells to reduce MTT [3-(4,5-dimethylthiozol-2yl)-2,5-diphenyl tetrazolium bromide] to formazan. Viability is expressed
as percent of control. *Significant difference ( p < 0.05) compared to control.
but in both instances the qualitative amounts of both were
reduced compared to parental cells (Figs. 3A and 4A). This
reduction was especially pronounced for the cleavage of
caspase-3. An analysis of LDH release into the growth medium
showed a release of 40–60% of total LDH from the cells under
conditions of Cdþ2 exposure that resulted in only a 50–60%
loss of cell viability (Fig. 5C), a significant increase compared
to the release of LDH by the parental cells (Fig. 5A).
Apoptosis and Necrosis of Asþ3-Transformed UROtsa
(URO-ASSC) Cells Exposed to Asþ3
The next goal of this study was to determine if UROtsa cells
malignantly transformed through exposure to Asþ3 would have
an alteration in the mechanism of Asþ3-induced cell death
compared to that of the parental cells exposed to Asþ3. It was
shown using DAPI staining that 9.6 ± 1.0% of URO-ASSC
cells exhibited profiles of condensed and fragmented nuclei
when exposed to 12lM Asþ3 for 30 h, features consistent with
cells undergoing apoptosis (Fig. 1D). DNA laddering and
caspase activation could be shown for the URO-ASSC cells
exposed to 16lM Asþ3 (Figs. 3D and 4D). An analysis of LDH
release into the growth medium showed a release of 30–40% of
total LDH from the cells under conditions of Asþ3 exposure
that resulted in a total loss of cell viability (Fig. 5D), a small but
significant increase compared to the release of LDH by the
parental cells (Fig. 5B).
þ3
As
AND Cd
þ2
-INDUCED UROTHELIAL CELL DEATH
297
FIG. 3. Agarose gel electrophoresis of DNA extracted from Cdþ2-and Asþ3-exposed parent and transformed UROtsa cells. (A) UROtsa parent cells exposed to
27lM Cdþ2. (B) UROtsa parent cells exposed to 16lM Asþ3. (C) UROtsa Cdþ2-transformed cells exposed to 27lM Cdþ2. (D) UROtsa Asþ3-transformed cells
exposed to 16lM Asþ3.
Expression of MT Protein in UROtsa, URO-ASSC, and
URO-CDSC Cells Exposed to Cdþ2 and Asþ3
The last goal of this study was to determine if metal induction of the MT protein was altered by the transformation of
the UROtsa cells. To determine this, the level of MT protein
was determined in UROtsa, URO-ASSC, and URO-CDSC cells
over a 48-h time course of exposure to either 12lM Cdþ2 or
8lM Asþ3. Lower doses of metals were used for this assay as at
high doses, total protein estimations in cell lysates were not
possible at 48 h due to extensive cell death. The results showed
that the levels of the MT-1/2 protein increased for both the
parental and URO-CDSC cells over the time course of exposure to Cdþ2; however, there was no difference in the level of
MT-1/2 protein accumulation between the parental and the
transformed cells (Fig. 6A). For the Asþ3 exposed cells, the results showed that the level of MT-1/2 protein did not increase
for either the parental or the ASSC cells over the time course of
exposure and that there was no difference in MT-1/2 protein
expression between the parental and Asþ3-transformed cells
(Fig. 6B). A corresponding analysis of the MT-3 protein
showed only background levels of MT-3 protein expression
in all three cell lines over the time course of exposure (data not
shown).
DISCUSSION
The first goal of this study was to determine the mechanism
of cell death when human urothelial cells were exposed to Asþ3
or Cdþ2. The results demonstrated that the mechanism of cell
death for UROtsa cells exposed to Asþ3 was primarily through
apoptosis, but there was also a significant contribution due to
necrotic cell death. That both mechanisms were operational in
Asþ3-induced cell death was determined using a combination
of qualitative and quantitative measurements. The presence of
apoptosis was confirmed by determining the existence of
fragmented nuclei upon DAPI-staining of cells, the existence
of a classic DNA ladder upon gel electrophoretic analysis, and
the detection of cleaved caspase-3 and caspase-9 products in
cell lysates by western analysis. These were all qualitative
measurements and indicated only that an appreciable number
of the cells were undergoing apoptotic cell death when exposed
to Asþ3. A quantitative measure of the amount of necrotic cell
death, and by extrapolation the amount of apoptotic cell death,
was determined by the analysis of LDH release into the growth
medium. This measurement is based on determining the
amount of necrotic cell death by release of the cytoplasmic
protein, LDH, into the growth medium. Specificity for necrosis
is based on the fact that necrosis is mediated by the rupture of
the cell membrane with release of cellular contents, while
apoptosis proceeds by the formation of membrane-bound
bodies which enclose the cytoplasmic contents which are
removed by phagocytosis. Using LDH release, it was determined that approximately 30% of the cells died by necrosis,
and from this it was assumed that the other 70% died by
apoptosis, when parental UROtsa cells were exposed to Asþ3.
There is only limited information on the effect of inorganic
arsenite on human urothelial cell death. Furthermore, extrapolation from animal models to humans is difficult since Asþ3
has been shown not to be carcinogenic in animal models,
including the urinary bladder (Kitchen, 2001). However, DMA
has been shown to serve as an effective substitute and can act as
either a promoter or a complete carcinogen in the rat bladder
(Life Sciences Research, 1989; Wanibuchi et al., 1996; Wei
et al., 1999; Yamamoto et al., 1995). Examination of the
urinary bladder using scanning electron microscopy of female
F344 rats administered 100 ppm DMA showed leafy microridges, extensive pitting, increased separation of epithelial
cells, exfoliation, and necrosis (Cohen et al., 2001; Shen et al.,
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FIG. 5. Release of LDH from parent and transformed UROtsa cells
exposed to Cdþ2 or Asþ3 for 36–48 h. (A) Parent and Cdþ2-transformed
UROtsa (URO-CDSC) cells were exposed to 27lM Cdþ2 for 36–48 h. (B)
Parent and Asþ3-transformed UROtsa (URO-ASSC) cells were exposed to
16lM Asþ3 for 36–48 h. The release of LDH was quantified spectrophotometrically at a wavelength of 490 nm. The results are expressed as percentage
of total LDH release. Total LDH was obtained by lysing untreated cells with
Triton X-100. *Significant difference ( p < 0.05) compared to parent cells.
FIG. 4. Western blot analysis for cleaved caspase-3 and -9 in parent and
transformed UROtsa cells. (A) Parent UROtsa cells exposed to 27lM Cdþ2. (B)
Parent UROtsa cells exposed to 16lM Asþ3. (C) UROtsa Cdþ2-transformed
cells exposed to 27lM Cdþ2. (D) UROtsa Asþ3-transformed cells exposed to
16lM Asþ3. Ten micrograms of total protein was subjected to electrophoresis.
Blots were also probed for actin to correct for protein loading.
2006). The technique of scanning electron microscopy was
particularly effective in detecting the necrotic lesions on the
bladder surface. This has led to the hypothesis that the necrotic
insult is followed by increased proliferation from tissue repair,
which leads to hyperplasia, and eventually the production of
a bladder tumor. The technique of scanning electron microscopy would not be expected to identify apoptotic cells; however, the observation of appreciable cell exfoliation would be
consistent with a concurrent apoptotic mechanism of cell
death. Furthermore, there has been one report detailing that
exposure to DMA does induce apoptosis in the rat urinary
bladder (Jia et al., 2004). To the author’s knowledge, there has
been no study that simultaneously detailed apoptosis and
necrosis in the bladder urothelium of rats exposed to DMA.
Thus, there is direct evidence that arsenic can elicit both
necrotic and apoptotic cell death of urothelial cells in the rat
bladder, and the findings using the UROtsa cells would be
consistent with these observations.
That Asþ3 can produce both necrosis and apoptosis of
urinary urothelial cells compliments recent studies which also
show that Asþ3 itself stimulates the specific cell signaling
transduction pathways involved in cell proliferation (Germolec
et al., 1996). Similar to classic tumor promoters, arsenic has
been shown to activate AP-1 and to induce the early response
genes c-fos, c-myc, and c-jun. The UROtsa cells have also been
shown to respond to Asþ3 by increasing their rate of cell
proliferation and AP-1 activity (Luster and Simeonova, 2004;
þ3
As
AND Cd
þ2
-INDUCED UROTHELIAL CELL DEATH
FIG. 6. Expression of MT-1/2 protein in parent and transformed UROtsa
cells treated with Cdþ2 or Asþ3 for 0–48 h. (A) UROtsa parent and Cdþ2
transformed (URO-CDSC) cells were exposed to 12lM Cdþ2 for 0–48 h. (B)
UROtsa parent and Asþ3 transformed (URO-ASSC) cells were exposed to 8lM
Asþ3 for 0–48 h. MT-1/2 protein levels were measured using an immunoblot
assay. *Significant difference ( p < 0.05) compared to untreated cells.
Simeonova et al., 2000). A dual role of Asþ3 in being able to
promote both cell death and cell proliferation in the normal
urothelial cell would be consistent with this laboratory’s
observations of the UROtsa cells during malignant transformation by Asþ3 (Sens et al., 2004). In this study, it was
shown that confluent cultures of UROtsa cells exposed to 1lM
Asþ3 underwent cell death only after being continuously
exposed to Asþ3 for over 30 days. This was followed by
regrowth of the few remaining viable cells to form a new
monolayer in the continued presence of the identical level of
Asþ3. This was then followed by a second instance of overt cell
toxicity where the remaining cells rapidly regrew to confluency
and displayed an increased rate of cell growth compared to the
parental cells. It was shown by the growth in soft agar and
heterotransplantation into nude mice that these resulting cells
had undergone malignant transformation. These observations
would be consistent with a mechanism of initial Asþ3-induced
toxicity followed by the subsequent stimulation of the cells
to proliferate once again to confluency. For this mechanism
to be operational would require the cells that survive initial
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Asþ3-induced toxicity to be more resistant to Asþ3. This is
exactly what was found in the present study when the parental
UROtsa cells were compared to the Asþ3-transformed UROtsa
cells, that is, the transformed cells were more resistant to Asþ3,
and the mechanism of cell death was shifted away from
apoptosis toward a necrotic mechanism of cell death. Thus,
under this expanded hypothesis, Asþ3-induced cytotoxicity can
set the stage for tissue repair in the urothelium and then subsequently act as a tumor promoter during the regeneration
process through the stimulation of the regrowth of cells that
have gained an increased resistance to Asþ3. In the transformation studies with the UROtsa cells, the concentration of
Asþ3 eliciting initial cell toxicity was the same as that which
allowed regrowth and transformation of the cells.
The mechanism underlying the increased resistance to Asþ3
that develops during malignant transformation of the UROtsa
cells is not known. The present study did examine MT
expression since it is known that MT can associate with Asþ3
and MT has been shown to be overexpressed in human archival
specimens of bladder cancer (Yamasaki et al., 2006; Zhou
et al., 2006a,b) and also in the tumor heterotransplants
generated from the Asþ3-transformed UROtsa cells (Zhou
et al., 2006a,b). However, it was shown that the basal and
induced expression of the MT protein was similar between the
parental and Asþ3-transformed cells, providing strong evidence
that MT was not a mediator of the increased resistance to Asþ3.
The results of the present study demonstrated that the
responses of the UROtsa cells to Cdþ2 were very similar to
those of the Asþ3 exposed cells. These responses included the
observation that Cdþ2 elicited both apoptotic and necrotic cell
death of the parental UROtsa cells, that apoptosis was mediated
by the caspase-3 pathway, and that the Cdþ2-transformed cells
had enhanced resistant to Cdþ2 and were more likely to
undergo necrotic cell death. It was also shown that MT was
unlikely to participate in the increased resistance noted for the
Cdþ2-transformed cells. There were also striking similarities
between the two metals in the past study which detailed the
malignant transformation of the UROtsa cells (Sens et al.,
2004). In this study, the concentrations of the metal used for
transformation were identical and the response of the cells to
exposure were very similar, with two episodes of cell death
followed by regrowth of the monolayer and subsequent
malignant transformation of the cells. The tumor heterotransplants were also similar in histology with the only difference
being that Asþ3-transformed heterotransplants displayed more
frequent areas of squamous differentiation. These similarities
can be used to argue that Cdþ2 should be examined more
aggressively as a possible risk factor in the development of
bladder cancer, either alone or in combination with Asþ3. As
noted in the introduction, there is a substantial body of research
that implicates arsenic in the development of human bladder
cancer. In contrast, even though Cdþ2 has also been evaluated
to be a serious human carcinogen it has not been as
aggressively examined for its possible role in bladder cancer.
300
SOMJI ET AL.
The present results would suggest that such an examination
might be warranted.
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ACKNOWLEDGMENTS
The project described was supported by grant number R01 CA094997 from
the National Cancer Institute (NCI), National Institutes of Health (NIH). Its
contents are solely the responsibility of the authors and do not necessarily
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