Download cell death associated with adenovirus mediated prodrug activation

cell death associated with adenovirus mediated
prodrug activation therapy
in HNSCC tumor model
Deepika Srivastava, Kumaravel Somasundaram*
and Rita Mulherkar#
Advanced Centre for Treatment, Research and Education in Cancer, Tata
Memorial Centre, Kharghar, Navi Mumbai, 410210 and *Microbiology and
Cell Biology, Indian Institute of Science, Bangalore, 560012, India
#corresponding author
Key words: adenovirus, prodrug activation, HNSCC, Ganciclovir, thymidine
kinase, apoptosis
Abstract
Adenoviral mediated suicide gene therapy has been shown to have
tumoricidal effect against a wide variety of tumor models. Even though the
validity of this approach has been confirmed, the molecular mechanism of
Adv-HSVtk/GCV induced cell death is still not well established. We examined
the mode of cell death by Adv-HSVtk/GCV in head and neck squamous cell
carcinoma cell line NT8e. Our results show that cell death caused is
independent of inter-nucleosomal DNA fragmentation or gene expressions
indicative of apoptosis. Moreover apoptosis was not evident from cell cycle
kinetic analysis. Adv-HSVtk/GV treated cells showed an accumulation of cells
in the S-phase of cell cycle, in a time dependent manner but there was no
increase in the “apoptotic peak” or the sub G1 population. Our results indicate
swelling of cytoplasm but chromatin condensation was not observed,
suggesting a possible involvement of necrosis. A nonapoptotic mechanism
may play a central role in the Adv-HSVtk/GCV-induced cell death in NT8e
cells.
1. Introduction
Oral cancer is one of the most common cancers in the world. In India, head
and neck (HN) cancers account for 30-40% cancers at all sites, out of which
9.4% being oral cancers. Squamous cell carcinoma (SCC) is the commonest
histological type comprising of 93.29% cases. Majority of HNSCC are
histologically squamous cell carcinoma affecting the age group 40-69 years
with males outnumbering females (2.9:1)(1) Treatment of HNSCC outcomes
remain consistently poor with 2-year survivals rarely exceeding 30% in
patients with advanced stage III and IV disease (2) Because of the dismal
results of conventional therapy, a new thrust in treatment strategies has
focused on the use of gene therapy.
One of the most promising strategies for gene therapy is the suicide gene
therapy. In this strategy, viral vectors containing metabolic enzyme genes
infect the target cells, and enzyme expression converts an inactive prodrug to
a toxic product. Upon viral transduction, the toxic product then selectively kills
target cells. One such prodrug system involves the introduction of herpes
simplex virus thymidine kinase gene (HSVtk) into tumor cells followed by the
administration of ganciclovir (GCV). HSVtk transduced cells selectively
convert GCV, a guanosine analogue, into a monophosphorylated form which
is then triphosphorylated by the endogenous kinases. Tk-GCV induces cell
death via chain termination after incorporation of GCV-triphosphate into
replicating DNA as well as inhibition of DNA polymerase alpha, thereby
inducing cell death (3). Another promising aspect of this strategy is the
“bystander effect” where the cytotoxic molecule (triphosphorylated GCV) is
transferred to adjacent untransduced cells via gap junctions thereby killing
them (4, 5).
The cytocidal efficacy of the Tk-GCV system in the treatment of various
tumors has been demonstrated both in vitro and in vivo {Ketola, 2004 #58;
Maatta, 2004 #61; Kwong, 2002 #5; Robe, 2004 #24}. Apoptosis has been
suggested as the cytocidal mechanism of Adv-HSVtk/GCV transduced cells
(9-12) A study on colorectal cancer (13) showed combination of apoptosis in
the G1 cell cycle phase and late apoptotic or necrotic sub-G1 DNA
fragmentation, depending on the tumor cell line, upon treatment with AdvHSVtk/GCV. Another study (14) showed that, GCV induced death of AdvHSVtk transduced oral SCC cells was mediated through an apoptotic
pathway. Contrary to the above, it was suggested that a nonapoptotic
mechanism may play a central role in the HSV-tk/GCV-induced cell death in
oral SCC cell lines (15). Even though the validity of this approach has been
confirmed, the molecular mechanism of Adv-HSVtk/GCV induced cell death is
still not well characterized. Therefore, in the present study we aim to
determine the mode of cell death by Adv-HSVtk/GCV induced cell death and
to verify whether apoptotic or non-apoptotic responses are associated with
Adv-HSVtk/GCV induced cell death in HNSCC cell line NT8e cells.
2. Materials and Methods
2.1. Cell lines, culture conditions and antibodies
HNSCC cell lin, NT8e was used for all the experiments. NT8e is derived from
pyriform fossa of a cancer patient and was developed and characterized in
our lab (16). HEK293 cells (human embryonic kidney cells) were used for
virus preparation and viral titer estimation. All cell lines were grown in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FCS
(Gibco, USA). The cells were maintained at 37°C in humidified air incubator
under 5% CO2 conditions. Caspase-3 antibody was obtained from Cell
Signaling Technology; β-tubulin antibody was obtained from Sigma-Aldrich.
2.2. Adenovirus vector preparation and titre determination
Recombinant Adv-HSVtk was prepared in our lab as described earlier
(Kothari et al 2010). For preparing the virus stocks, 3x106 HEK293 cells were
cultured in 20x100mm plates. These cells were then transduced with enough
viruses so that they demonstrate cytopathic effect (CPE) within 36hrs.
Infected cells and media were pooled and centrifuged at 1400 rpm for 10
minutes. The cell pellet was resuspended in 100mM Tris (pH 8) and lysed by
rapid freeze-thaw. Lysate was centrifuged and the supernatant was overlaid
on a CsCl density gradient and centrifuged at 32000g for 1 hr. The lower virus
band was collected and mixed with 2x glycerol buffer and stored at -20°C. To
determine the tier of adenovirus stock the method of Gueret et.al (17) was
followed. In brief, 5x105 HEK293 cells were plated in a 6 well dish. Cells were
infected with Adv-HSVtk at various dilutions, harvested 17-20 hrs post
infection and analyzed for GFP+ cells by Flow cytometry. Assumption made is
that one fluorescent cell is infected by a single infectious particle at a
relatively low Multiplicity of Infection (MOI). At low MOI a linear relationship
between the proportion of infected cells and the dilution factor can be drawn.
The titre was reported as green fluorescent units per ml (gfu/ml) and was
calculated using the following formula:
Titre (gfu/ml) = % INF x Total cell no. x Dil
100 x Vol
Explain all the short forms
Titre of ~1x109 gfu/ml was achieved.
2.3. Cell viability assay: Sulpho Rhodamine B (SRB) assay
NT8e cells were seeded in a 96 well plate. Next day Adv-HSVtk at various
dilutions was added to each well. 6 hrs post adenoviral transduction, GCV at
a concentration of 0, 0.5, 1 and 2 μg/ml was added to the corresponding
wells. Cell viability was assessed 72 hrs after GCV treatment using SRB
assay (18). Briefly, cells were overlaid with 100 µl of 50% TCA and incubated
at 4°C for 1.5 hrs. The cells were washed with water and allowed to air dry.
50 µl SRB dye was added to each well and the plate was incubated in dark
for half hour. The plate was then washed with 1% acetic acid and air dried.
The bound dye was solubilised using 10 mM Tris pH 10.5 and absorbance
was taken at 540nm. Cell viability was expressed as relative percent to
control. The results represent the mean + SE of triplicates.
2.4. Confocal microscopy DAPI staining
NT8e cells were seeded in a 35 mm plate and were infected with 1x105 gfu
Adv-HSVtk followed by 2µg/ml GCV treatment for 72 hrs. After the end of the
treatment duration, cells were washed with 1X PBS and fixed in 4% PFA for
10 mins at RT. Cells were stained with 10 µg/ml DAPI for 1 min followed by
three washes with 1X PBS and acquired on Confocal microscope.
2.5. Acridine orange/Ethidium bromide staining
NT8e cells were seeded in a 35 mm plate and were infected with 1x105 gfu
Adv-HSVtk followed by 2 µg/ml GCV treatment for 72 hrs. After the end of the
treatment duration cells were washed with 1X PBS and Acridine Orange
/Ethidium Bromide to a final concentration of 1 mg/ml was added to each
plate and cells were observed under an inverted microscope.
2.6. Cell cycle kinetics
To quantitate the percent apoptosis and to distinguish populations of cells in
different phases of cell cycle, by flow cytometry, NT8e cells were treated with
1x105 and 1x106 gfu Adv-HSVtk and 2µg/ml GCV. Cells were harvested on 0
hr, day - 1, 2 and 3. Corresponding NT8e untreated controls of 0hr, day1, 2
and 3 were harvested. Cells were fixed with 70% ethanol, stained with
4mg/ml PI and acquired on FACS Calibur (BD Biosciences, USA).
2.7. DNA ladder assay
NT8e cells were seeded in a 60 mm plate and were infected with 1x105 and
1x106 gfu Adv-HSVtk followed by 2 µg/ml GCV treatment for 72 hrs. The DNA
Ladder assay was performed as reported previously (19). Briefly, on day 3
cells were harvested and treated with incubation buffer (10mM Tris pH 7.4, 10
mM EDTA, 0.5% Triton-X100) to remove cell debris and genomic DNA. The
supernatant was treated with RNase (Sigma, 1mg/ml) and proteinase-K
(Sigma, 20 mg/ml) for 2 hrs at RT. DNA was extracted using isopropanol
precipitation. The pellet was dissolved in TE and run on a 2% agarose gel at
100V for 1 hour 30 mins. NIH3T3 cells treated with 100nM staurosporine
(Sigma) for 24hrs was taken as positive control. Use NT8e with St and show
ladder or delete this part from the ms
2.8. Western blotting
NT8e cells were treated with Adv-HSVtk/GCV for different time point’s viz. 2
hrs 6hrs, 12 hrs, 18 hrs, 24 hrs, 36 hrs, 48 hrs and 72 hrs. Each time point
lysate was made, resuspended in Laemmli’s sample buffer, boiled, and
resolved on 12% SDS-PAGE. Proteins were transferred onto polyvinylidene
difluoride (PVDF membrane – Amersham, USA) membrane and the
membrane was probed with 1: 500 dilution of anti-caspase-3 antibody (Cell
Signaling). The blots were developed by using the ECL Plus system
(Amersham Biosciences). The blot was stripped using buffer (Thermo
Scientific) and β-tubulin was used for equal loading (1:1000, Sigma Aldrich).
2.9. Quantitative Real time PCR for 96 apoptotic genes
RNA was extracted from NT8e cells treated with Adv-HSVtk/GCV for 72 hours
using TRIzol Reagent (Invitrogen). The cDNA was made using the
Superscript First strand synthesis kit (Invitrogen). 10 ng cDNA was used for
real time PCR. A real time PCR array was done using the method patented by
KS. Expression analysis for 96 apoptotic genes was studied and fold change
with respect to untreated control NT8e was reported.
3. Results
3.1. Adv-HSVtk/GCV mediated cell death in NT8e cells
To elucidate the mechanism of cell kill by Adv-HSVtk/GCV system on NT8e
cells, the effect of GCV on cell viability, cell cycle kinetics, DNA fragmentation,
nuclear condensation and apoptotic gene expression were studied. Our data
demonstrates that GCV inhibited the growth of Adv-HSVtk transduced NT8e
cells in a dose dependent manner (Fig 1). A concentration of 2µg/ml GCV
reduced survival to almost 50% when 0.25µl or 0.125µl of the stock virus
(which corresponds to 2.5x105 and 1.25x105 gfu respectively) was used for
transduction. There was minimal nonspecific toxicity by GCV as shown by the
untransduced NT8e cells. Therefore in subsequent experiments, NT8e cells
were treated with 1x105 gfu Adv-HSVtk and 2µg/ml GCV for three days.
3.2. Absence of nuclear condensation as observed by DAPI
Fluorescent microscopic studies using DAPI staining revealed that the size of
nuclei of cells treated with GCV for 3 days was larger than that of the control
untreated cells but nuclear fragmentation and chromatin condensation
characteristic to apoptosis were not recognized in cells treated with Adv-
HSVtk/GCV for a period of 3 days (Fig 2). Likewise AO/EB also revealed
necrotic cells (Fig 3) upon treatment with Adv-HSVtk/GCV for three days.
Acridine orange (AO) permeates all cells and stains the nuclei green.
Ethidium bromide (EB) is only taken up by cells when cytoplasmic membrane
integrity is lost, and stains the nucleus red. EB also dominates over AO
therefore, live cells show green stained nuclei with organized structures;
apoptotic cells contain condensed or fragmented chromatin (green or
orange); necrotic cells stain orange but with a nuclear morphology similar to
that of viable cells.
3.3. Increase in the S-phase population without an apparent sub-G1
population of NT8e cells upon treatment with Adv-HSVtk/GCV
Flow cytometry was performed as mentioned in the materials and methods, to
distinguish population of cells in different phase of cell cycle and also in
apoptosis. Flow cytometry of propidium iodide stained cells revealed that cells
treated Adv-HSVtk/GCV for a period of 3 days, showed a gradual increase in
the proportion of cells in the S phase of cell cycle (Fig 4, Table 1). At 0 hr 22%
of cells were in S phase but the proportion of S phase cells for day 1, day 2
and day 3 became 42, 71 and 65% resp. upon Adv-HSVtk/GCV treatment.
Apoptotic cells were not observed upon treatment with Adv-HSVtk/GCV. Even
after 3 days of Adv-HSVtk/GCV treatment only 9% of apoptotic cells were
observed.
3.4. Absence of DNA fragmentation upon Adv-HSVtk/GCV treatment
Appearance of DNA fragments or laddering of cellular DNA is used as a
marker for apoptosis. Agarose gel electrophoresis of DNA demonstrated that
the DNA ladder characteristic to apoptosis was not observed in NT8e cells
treated with 1x105 and 1x106 gfu Adv-HSVtk and 2µg/ml GCV for three days
(Fig 5). Cellular DNA preparations from untreated NT8e cells, which were
included as the negative control, did not display DNA ladder pattern.
However, the positive control i.e. 100nM staurosporine treated NIH3T3 cells,
showed a distinctive DNA ladder pattern.
3.5. Active or cleaved caspase-3 band undetectable upon Adv-HSVtk/GCV
treatment
Western blot analysis did not show activation of caspase-3 (cleaved caspase3 band at ~17KD) on Adv-HSVtk/GCV treatment for different time points.
However, the positive control, 100nM staurosporine treated NIH3T3 cells
showed a distinctive 17KD band (Fig 7).
3.6. Quantitative Real Time PCR array analysis of several apoptotic genes
Real Time quantitative analysis for 96 different apoptotic genes was
performed (Fig 6). Fold increase with respect to the level of the gene
expression in untreated NT8e control was considered. Fold increase of 2 or
more was taken as indicative of differential gene expression. Some of the
important genes involved in apoptosis like caspase-9, caspase-7, FADD,
BAK1, BID etc did not show any differential expression with respect to
untreated control. However, pro-apoptotic genes like BIK, BNIP and NOXA
showed approximately 2 fold up regulation while pro-apoptotic genes like LTA
and TNFRSF9 showed approximately 3 fold up regulation. Anti-apoptotic
genes like NOL3, Bcl2 and BIRC1 showed 3 to 4 fold down regulation.
4. Discussion
In the present study we have used NT8e cells line as a model system for
HNSCC to investigate the mode of cell death by adenovirus mediated
prodrug activation therapy. As is evident from SRB assay, NT8e cells
transduced with Adv-HSVtk show susceptibility to GCV in a dose dependent
manner showing no toxicity to untransduced NT8e cells. A survey of literature
reveals that while some groups suggest induction of apoptosis on AdvHSVtk/GCV treatment (10, 20, 21), there are contrary reports to indicate
involvement of non apoptotic cell death upon the same treatment (22, 23). To
investigate the mode of cell death in Adv-HSVtk/GCV transduced NT8e cells;
we performed classical DAPI and Acridine orange/Ethidium bromide (AO/EB)
staining experiments. Surprisingly, no nuclear condensation in AdvHSVtk/GCV transduced NT8e cells was observed when the cells were
stained with DAPI while, AO/EB staining indicated presence of necrotic cells
(red labeled cells). However in a previous report by Pei-Xiang et al. (1999),
AO/EB staining revealed morphological features characteristic of apoptosis in
Adv-HSVtk/GCV treated MDA-MB-468 and MCF-7 cells (24)
To further elucidate the mechanism of cell death, we did flow cytometric
analysis of Adv-HSVtk/GCV treated NT8e cells. Adv-HSVtk/GCV induces cell
death via chain termination after incorporation of GCV-triphosphate into
replicating DNA as well as inhibition of DNA polymerase alpha thus leading to
a gradual increase of the proportion of cells in S phase, in keeping with the
known mechanism of action of GCV (3). Our flow cytometric data indicated an
increase significant? P<? in the proportion of cells in the S-phase of cell cycle
upon Adv-HSVtk/GCV treatment of NT8e cells. Further, in accordance with
our previous data no sub G1 fraction “apoptotic peak” was observed. Earlier
studies of flow cytometry by Sung-Jen Wei et al (1998) (25) demonstrated
that HSV-tk/GCV treatment caused either S- and/or G2/M-phase cell cycle
arrest before undergoing cell death. They showed an increase and
accumulation of S-phase B16F10 melanoma cells upon HSVtk/GCV
transduction along with a prominent increase in the apoptotic population at 72
hrs. However, a report by Kuratate et al (2005) showed cell death progressing
with no cell cycle arrest (15)
To further confirm our observations DNA ladder assay was carried out which
is considered to be one of the reliable methods to detect apoptosis. Samejima
et al (1995) and Sung-Jen Wei et al. (1998), (25, 26) have independently
reported a typical apoptotic DNA ladder pattern upon treatment with
HSVtk/GCV. Sung-Jen Wei et al (1998) (25) also clearly showed DNA ladder
pattern in HSVtk treated B16F10/TK-C19 cells treated with GCV. Yoshiyasu et
al (1995) (23) also did not observe apoptotic DNA ladder and concluded that
apoptosis was not involved in XCtkn2, GCV treated hepatoma cells. Similar to
such reports, we did not observe any apoptotic DNA ladder when NT8e cells
were transduced with Adv-HSVtk/GCV. Even on increasing the virus
concentration to 1x106 gfu Adv-HSVtk we could not detect DNA ladder like
pattern.
Caspase-3 is one of the key effector caspases in the cell. On activation,
procaspase is cleaved into a lower molecular weight protein which further
cleaves other protein substrates to trigger the apoptotic process. A ~17KD
band corresponding to cleaved caspase could not be detected in AdvHSVtk/GCV transduced NT8e cells. Likewise, Kuratate et al (2005) have also
reported that upon HSVtk/GCV treatment no cleaved or activated caspase-3
band is observed (15). All this data suggests that some alternative
mechanism, other than apoptosis might be playing a role in the cytocidal
effect of HSVtk/GCV treatment on NT8e cells.
To get an insight into the molecular mechanism of the cell death induced by
Adv-HSVtk/GCV in NT8e cells we performed a real time PCR array for 96
genes involved in apoptosis. Our real time data does not reveal differential
expression of any significant genes involved in apoptosis. Key players of
apoptosis like caspase 9, caspase-1, FADD and BID did not show any
differential regulation upon Adv-HSVtk/GCV transduction in NT8e cells.
However, a down regulation in some anti apoptotic genes like NOL3 and
BIRC1 was observed. Also some pro-apoptotic genes like NOXA and BNIP3
were up regulated which are reported to be involved in p53 mediated
apoptosis (27, 28) and hypoxia (29) respectively. Despite the differential
expression of these genes apoptosis is not observed suggesting that their
differential expression may not be sufficient to induce apoptosis in AdvHSVtk/GCV transduced NT8e cells.
Our report contradicts earlier published reports which establish apoptosis as
the mode of cell death upon Adv-HSVtk/GCV transduction. It is known that
same stimuli induce apoptosis and necrosis both under different conditions
(30). TNF is one such molecule which is known to induce apoptosis or
necrosis depending on the type of cell line used (31). We have used simple
and basic approach to understand the mode of cell death in NT8e cells upon
Adv-HSVtk/GCV transduction. Our preliminary data clearly suggests that AdvHSVtk/GCV treatment induces cell death in NT8e cells but apoptosis may not
be the central pathway through which cell death occurs. The triggering
mechanisms of apoptotic and non-apoptotic cell death pathways may differ
depending on the type of cell lines. But the exact reason for the discrepancy
between the previous reports and the present study needs to be further
investigated.