Re-engineered p53 Activates Apoptosis In Vivo and Causes Primary Tumor Regression in A Dominant Negative Breast Cancer Xenograft Model

Inactivation of p53 pathway is reported in more than half of all human tumors and can be correlated to malignant development. Missense mutation in the DNA binding region (DBD) of p53 is the most common mechanism of p53 inactivation in cancer cells. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain (TD). Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have previously designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain capable of escaping transdominant inhibition by mutant p53 in vitro. In this report, we demonstrate the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model in vivo. In addition, we illustrate the profound effects of the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells. Finally, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model.


INTRODUCTION
The ability of p53 to achieve tumor suppressor function depends on formation of p53 tetramers to act as a transcription factor of several target genes. 1,2 Once activated, p53 stimulates a wide network of signals including DNA repair, cell cycle arrest, and apoptosis. 3 The significance of p53 function is highlighted by the correlation of its inactivity and malignant development. Inactivation of p53 pathway is reported in more than half of all human tumors and can be achieved via several mechanisms including nuclear exclusion and hyperactivation of MDM2, the main regulator of p53 function. [4][5][6] However, acquisition of missense mutations in one or both alleles of the TP53 gene remains the most common mechanism of p53 inactivation. 7 The majority of these mutations take place in the DNA binding domain (DBD) which is responsible for p53 interaction with DNA. Although mutant p53 in cancer cells may have impaired tumor suppressor function and transcriptional activity, it retains its ability to oligomerize with other mutant or wild-type (wt) p53 via the tetramerization domain (TD). 8,9 When mutant p53 oligomerizes with wt-p53 through hetero-oligomerization, the resulting tetramer has impaired function in most cases due to transdominant inhibition by mutant p53 (Figure 1). The outcome of this transdominant inhibition varies significantly based on the type of mutant p53 present in cells. 10 This phenomenon is known as the dominant negative effect of mutant p53 and gives rise to a critical barrier to utilizing wt-p53 for cancer gene therapy. 11 Our goal was to design a new, chimeric superactive p53 with the following activity: wt-p53 like functional transcriptional activity; promotion of improved, highly potent p53-dependent apoptosis; and circumvention of the dominant negative inactivating effect of endogenous mutant p53 in cancer cells. To this end, we engineered a chimeric p53 (p53-CC) that has an alternative tetramerization domain and showed its ability to escape transdominant inhibition by mutant p53 in vitro. 12 The Bcr oligomerization domain is a 72 amino acid coiled-coil (CC) motif that serves as an alternative oligomerization domain of p53-CC to evade heterooligomerization with endogenous mutant p53, and hence, bypass the dominant negative effect reported in cancer cells. The CC domain itself was tested as a control previously, and was found to be non-toxic. 12 p53-CC activity was found to retain similar tumor suppressor activity compared to exogenous wt-p53 in several cancer cell lines harboring different p53 statuses (null, wt, wt mislocalized, and mutant non-dominant). Finally, we investigated potential transdominant inhibition of p53-CC and wt-p53 via co-expression of a potent dominant negative mutant p53. As hypothesized, p53-CC retained the same levels of activity regardless of the presence of transdominant mutant p53, while wt-p53 showed loss of activity. 12 In this report, we demonstrate the superior tumor suppressor activity of p53-CC in vitro and in vivo in MDA-MB-468, an aggressive p53-dominant negative breast cancer cell line. Furthermore, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 in the MDA-MB-468 tumor model. Our viral-mediated gene therapy approach succeeds in demonstrating the effects of the transdominant effect of endogenous mutant p53 over p53-CC and wt-p53.

p53-CC induces higher levels of cell death compared to wt-p53
We 12 and others 13,14 have shown that the MDA-MB-468 human breast adenocarcinoma cell line serves as a suitable dominant negative mutant p53 model for testing the effect of p53-CC and wt-p53. The endogenous p53 in MDA-MB-468 contains the R273H point mutation, which is known to exhibit a dominant negative effect over wt-p53. 12,14 As we have shown previously 12,15 , p53-CC is capable of inducing cell death in this as well as other cancer cell lines, regardless of endogenous p53 status. Figure 2 illustrates the superior tumor suppressor function of p53-CC over wt-p53 in a 7-AAD viability assay which stains apoptotic and necrotic cells 16,17 (compare Figure 2A vs 2B). Wt-p53 activity is not significantly different from that achieved by the negative control Ad-ZsGreen1 ( Figure 2B vs 2C). This observation illustrates the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells and highlights the significance of our approach to escape transdominant inhibition. These results are summarized in Figure 2D. Preceding animal studies, we explored the mechanism of cell death, and hypothesized that it occurs via an apoptotic pathway. Thus, three different apoptosis assays: the tetramethylrhodamine ethyl ester (TMRE) assay (analogous to the JC-1 assay), activated caspase-3/7 assay, and annexin-V staining were carried out.

p53-CC caused cell death via the apoptotic pathway
Mitochondrial depolarization is measured by loss in TMRE intensity correlates with an increase in mitochondrial outer membrane permeabilization (MOMP). 18 TMRE is a cationic, cell-permeant, and fluorescent dye that rapidly accumulates in mitochondria of living cells due to the negative mitochondrial membrane potential (ΔΨm) of intact mitochondria compared to cytosol. 19,20 Mitochondrial depolarization results in loss of TMRE from mitochondria and a decrease in mitochondrial fluorescence intensity (FI) 7 , illustrated as %MOMP induction in Figure 3A. Figure 3A demonstrates that p53-CC induced significantly higher levels of mitochondrial membrane permeabilization, a hallmark of intrinsic apoptosis, compared to wt-p53. Wt-p53 also induced mitochondrial membrane permeabilization, although not to the same extent as p53-CC. MOMP indicates that cells are transitioning to an apoptotic state. 21 To further investigate the potential apoptotic activity of p53-CC and wt-p53, we carried out a flow cytometry-based assay to detect the levels of activated caspase-3/7 in MDA-MB-468 cells ( Figure 3B). Caspase-3/7 activation is downstream from mitochondrial outer membrane permeabilization in the intrinsic apoptotic pathway and plays a central role at the execution-phase of cell apoptosis. [22][23][24] Figure 3B shows that cells treated with p53-CC display increased levels of active caspase-3/7 compared to those treated with wt-p53 or the negative control Ad-ZsGreen1.
Finally, annexin-V staining was performed, which measures externalization of phosphatidylserine on the cell surface of apoptotic cells specifically. 25,26 Figure 3C shows higher levels of annexin-V positive staining in cells treated with Ad-p53-CC compared to Ad-wt-p53; wt-p53 apoptotic activity was not significantly different from the negative control Ad-ZsGreen1. Cellular apoptosis as indicated in Figure 3C parallel the results from the 7-AAD staining in Figure 2.
To summarize, Figures 3 A, B, and C show MDA-MB-468 cells treated with Ad-p53-CC undergo significant apoptosis, validating p53-CC as a potent candidate for gene therapy. The levels of apoptosis induced by Ad-p53-CC are statistically significant compared to that of Ad-wt-p53 or Ad-ZsGreen1 in all three apoptosis assays ( Figure 3) in addition to the 7-AAD assay ( Figure 2).

In vivo efficacy in a dominant negative breast cancer animal model
MDA-MB-468 human breast adenocarcinoma represents an aggressive breast cancer cell line characterized as triple negative due to the absence of molecular targets including estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. 27 In addition, MDA-MB-468 cells harbor a dominant negative mutant p53 capable of impairing the function of wt-p53. [12][13][14] We therefore used this cell line to induce orthotopic breast tumors in mice to compare the impact of the dominant negative effect of mutant p53 on the biological activity of p53-CC and wt-p53 in viral-mediated gene therapy. Because of the presence of the coxsackievirus and adenovirus receptor (CAR), MDA-MB-468 cells can be transfected by adenovirus. 28 Induced in the mammary fat pad of female athymic nu/nu mice, MDA-MB-468 tumors orthotopic engraftment fosters tumorigenesis to occur in the appropriate macro-as well as microenvironment mimicking the environment of human MDA-MB-468 tumors. 29,30 Due to this, MDA-MB-468 is a commonly used xenograft model for triple negative breast cancer. 31,32 Tumors were allowed to grow to approximately 50 mm 3 prior to randomization of treatment groups which received intratumoral injections of Ad-p53-CC or Ad-wt-p53. The empty viral vector (Ad-ZsGreen1) served as a negative control in addition to an untreated control. Injections were made on days 0-4 and 7-11 for optimal efficacy 33 and consisted of 5.0×10 8 PFU of the viral constructs in a 50 µl volume. All procedures were performed according to established NIH guidelines and followed University of Utah Institutional Animal Care and Use Committee approved protocols. Figure 4A shows a representative image of a tumor bearing mouse with the mammary tumor located in the right inguinal area, while Figure 4B shows images of representative excised tumors from each treatment group. The tumor size reduction expected with these treatments served as a direct measure of the tumor suppressor function of our p53 variants. As expected, the Ad-p53-CC treatment group achieved statistically significant (p < 0.001) reduction in mean tumor size compared to Ad-wt-p53, Ad-ZsGreen1, and untreated groups ( Figure 4C). Although tumor reduction induced by Ad-wt-p53 is not statistically significant compared to the Ad-ZsGreen1 or untreated groups, Ad-wt-p53 treatment resulted in stable disease, halting tumor progression. The findings from Figure 4C reveal that p53-CC can achieve tumor regression of an aggressive p53-dominant negative breast cancer model in vivo, while wt-p53 is only capable of halting tumor progression. In addition, the excised tumors from the Ad-ZsGreen1 negative control and untreated groups appeared to be more vascularized compared to tumors derived from the treatment groups Ad-p53-CC and Ad-wt-p53 ( Figure 4B), potentially implying an additional anti-angiogenic effect of p53-CC and wt-p53 in vivo.
Animal body weights were regularly monitored throughout the study and no significant weight loss in animals was observed in any of the groups ( Figure 4D) rendering the treatment as well as the viral carrier safe. Throughout the study, the Ad-p53-CC treatment group maintained the smallest mean tumor size compared to all other groups. Both control groups (Ad-ZsGreen1 and untreated) exhibited the largest mean tumor size compared to all other groups throughout the entire study.

Histopathological evaluation of tumor tissues and evidence for tumor suppressor activity
The tumor size reduction observed in Figure 4C indicates tumor suppressor functionality of Ad-p53-CC as well as Ad-wt-p53 in vivo. To verify if this activity is indeed p53-dependent, we carried out immunohistochemical staining of p21 as it is one of the best characterized bona fide p53 target genes. 34 Photomicrographs of representative sections from harvested tumor tissues from each group are displayed in Figure 5A. The left column in Figure 5A exhibits hematoxylin and eosin (H&E) staining, while the middle column represents p21 immunohistochemical staining for each group. In addition, the right column in Figure 5A shows the intratumoral expression of our gene load (i.e. p53-CC or wt-p53) as a function of the ZsGreen1 fluorescent protein co-expressed with our genes of interest. Microscopic examination of H&E staining revealed higher levels of necrosis (solid arrows, necrosis; open arrows, non-necrotic areas) in all tumors harvested from mice injected with Ad-p53-CC compared to the Ad-wt-p53, Ad-ZsGreen1, or untreated groups ( Figure 5A). This implies the detected necrosis may be due to the tumor suppressor activity of p53-CC since the tumors did not reach a large enough size to develop a necrotic core, and as such the observed necrosis was due to treatment, not hypoxia.
3,3' diaminobenzidine (DAP) stains the nuclei of p21-positive cells brown, as shown in photomicrographs (middle column, Figure 5A). Unexpectedly, p21 immunohistochemistry staining revealed higher levels of p21 induction in the Ad-wt-p53 treatment group compared to the Ad-p53-CC treatment group. p21 is one of the key factors by which p53 enforces cell cycle arrest. The induction of cell cycle arrest by p21 converges with findings from Figure  4C where tumors from the Ad-wt-p53 treatment group show a halt (arrest) in tumor growth. As expected, p21 expression was not detected in the Ad-ZsGreen1 negative control or untreated groups, which validates that p21 expression is linked to direct p53 activation. Similar expression of ZsGreen1 across the different groups (Ad-p53-CC, Ad-wt-p53, and Ad-ZsGreen1) in the right column of Figure 5A indicates comparable intratumoral expression of our viral constructs. In addition, p53 immunohistochemistry staining was performed and equal levels of total (i.e. endogenous and exogenous) p53 expression were detected across all groups, including the untreated group, which relates to the known presence of endogenous p53 in MDA-MB-468 cells (data not shown). Figures 5 B and C represent semi-quantitative histoscore analyses of tumor necrosis and p21 up-regulation in the excised tumors from all groups.
Tissues from additional organs (liver, kidney, spleen, heart, and lungs) harvested from animals of all treatment groups showed normal physiology and no abnormalities or signs of pathology (data not shown). However, metastases of tumor cells to the gastrointestinal region were noted in two out of six mice in the Ad-ZsGreen1 group and three out of six mice in the untreated group (data not shown). This may imply an anti-metastatic function of p53-CC as well as wt-p53 in this tumor model although further examination is necessary.

Detection of pathway-specific markers for cell cycle arrest and apoptosis
Based on our observations, we postulate that p53-CC is capable of causing tumor size reduction in vivo by favoring the apoptotic pathway, while wt-p53 activity is biased towards inducing cell cycle arrest. To further investigate this hypothesis, we carried out immunoblotting of cleaved (activated) caspase-3 and p21 on samples from in vitro and in vivo. It is well known that all apoptotic pathways converge on caspase-3 (the main executioner caspase), 35,36 whereas p21 induction by p53 causes cells to undergo cell cycle G 1 phase arrest. [37][38][39] Therefore, detection of activated caspase-3 and p21 are acceptable biomarkers for apoptosis and cell cycle arrest, respectively. Figure 6A  Part of the excised tumor tissues from each animal was homogenized and lysed for western blotting. Figure 6D shows representative western blotting of MDA-MB-468 in vivo tumor tissue lysates. Similar to the findings obtained from in vitro western blotting ( Figures 6A-C), tumor tissues from the Ad-p53-CC treatment group showed lower p21 expression ( Figure 6E) but higher caspase-3 induction ( Figure 6F).
To further validate our hypothesis about the differential ability of p53-CC and wt-p53 to induce p21-depndent cell cycle arrest, we carried out cell cycle arrest evaluation via DNA content analysis ( Figure

DISCUSSION
The data obtained in this report supports our hypothesis that chimeric p53-CC has superior tumor suppressor function compared to wt-p53 in vitro and in vivo using a dominant negative mutant p53 model. Although the concept of a "superactive" p53 was reported in 2010, 40 there are no known reports of constructing a p53 capable of bypassing the dominant negative effect of mutant p53 in cancer cells and increases apoptosis (over wt-p53). p53-CC induces higher levels of cell death in vitro compared to wt-p53 in the 7-AAD assay ( Figure  2) as well as in the apoptosis assays: TMRE, caspase-3/7, and annexin-V (Figure 3). To validate if the superior activity of p53-CC in vitro translates in vivo, we carried out animal studies using an orthotopic MDA-MB-468 xenograft breast cancer model in mouse mammary fat pads. Indeed, intratumoral injections with Ad-p53-CC achieved substantial tumor regression that is statistically significant compared to the Ad-wt-p53, Ad-ZsGreen1, and untreated groups ( Figure 4C), without any sign of treatment toxicity ( Figure 4D). H&E staining of tumor tissues revealed higher levels of necrosis in all tumor tissues from mice injected with Ad-p53-CC compared to the Ad-wt-p53, Ad-ZsGreen1, or untreated groups. To test if the observed tumor suppressor activity of p53-CC in vivo is p53-dependent, immunohistochemistry staining of p21, the most well studied p53 target gene, 34 was conducted. As expected, p21-positive staining was observed only in the Ad-p53-CC and Adwt-p53 treatment groups ( Figure 5A) with higher p21 staining with Ad-wt-p53 treatment.
Since p53-CC was able to induce apoptosis (including caspase 3/7), and wt-p53 increased p21 expression, we explored a possible differential mechanism of p53-CC (favoring apoptosis) and wt-p53 (favoring cell cycle arrest) in MDA-MB-468 cells. To test this premise, immunoblotting was carried out on samples from in vitro (Figures 6A-C) and in vivo ( Figures 6D-F) to detect expression levels of p21, which induces cell cycle arrest, and caspase-3, a major executer of apoptosis. Figure 6 revealed that tumor tissues treated with Ad-p53-CC expressed low levels of p21 (reduced cell cycle arrest) but high levels of active caspase-3 (increased apoptosis). In contrast, tumor tissues injected with Ad-wt-p53 expressed high levels of p21 (increased cell cycle arrest) but low levels of caspase-3 (decreased apoptosis).
While Figure 3A-C validated that p53-CC induces higher levels of apoptosis compared to wt-p53, our cell cycle arrest analysis ( Figure 7A-C) revealed that wt-p53 induced higher levels of G0/G1 cell cycle arrest compared to p53-CC. These results may provide evidence that p53-CC and wt-p53 may have different mechanism of activity in the MDA-MB-468 breast cancer model.
The transdominant mutant p53 found endogenously in MDA-MB-468 cells retains the ability to hetero-oligomerize with exogenous wt-p53, since its tetramerization domain remains intact. 10 We and others have shown previously that upon hetero-oligomer formation, the activity of exogenous wt-p53 is impaired due to the dominant negative effect of mutant p53 in cancer cells. [12][13][14] Our chimeric p53-CC was designed to overcome this barrier with a use of an alternative oligomerization domain, a coiled-coil from Bcr (CC). This CC is known to tetramerize as an antiparallel dimer of dimers, similar to the tetramerization domain of wt-p53. 12,41 The use of this alternative oligomerization domain allows p53-CC to escape any possible hetero-oligomerization with mutant p53 and consequent transdominant inhibition. Indeed, our previous work validated the ability of p53-CC to exclusively form homo-oligomers. From a gene therapy point of view, the ability of p53-CC to evade transdominant inhibition gives it an advantage over wt-p53 in dominant mutant p53 cancer cells such as MDA-MB-468.
Our viral-mediated gene therapy in vivo studies show that p53-CC has superior tumor suppressor activity compared to wt-p53 in the MDA-MB-468 aggressive p53-dominant negative breast cancer model. In fact, p53-CC was capable of achieving significant tumor regression, while wt-p53 is only capable of halting tumor progression ( Figure 4C). Upon further investigation, we discovered that the difference in outcome of the tumor size reduction was due to the ability of p53-CC to activate the apoptotic pathway, whereas wt-p53 activates cell cycle arrest via p21 induction (Figure 8). These findings are supported by western blot analyses from in vitro ( Figure 6A-C) and in vivo ( Figure 6D-F) MDA-MB-468 cells/tumors, as well as cell cycle arrest analysis ( Figure 7A-C).
Analysis of p53-regulated gene expression patterns may possibly offer an explanation for differential pathway activation between p53-CC and wt-p53 (apoptosis vs cell cycle arrest). It has been shown that p53-responsive gene expression patterns are highly variable, depending on the p53 protein levels in the cell. 42 It is also known that higher levels of active p53 lead to activation of apoptotic genes, while lower levels of p53 activate cell cycle regulator genes. 43 In cells treated with p53-CC vs wt-p53, higher levels of the chimeric p53-CC protein exist compared to levels of active wt-p53 protein in cells (although they have similar expression levels), due to the ability of p53-CC to escape sequestration by mutant p53. Unlike p53-CC, substantial amounts of the wt-p53 protein are forced into inactive hetero-oligomers with endogenous mutant p53 (the dominant negative effect). This reduction in 'available' active wt-p53 could lead to failure in binding promoters of apoptotic genes that require higher active p53 protein levels in the cell. Cell cycle regulator genes, such as p21, would be activated instead, since wt-p53 possess higher binding affinities to these promoters (i.e. requires less p53 to bind and activate). In contrast, abundance in active chimeric p53-CC protein levels is found in cells treated with p53-CC (due to escaping transdominant inactivation and not difference in expression levels), which would lead to binding and activation of apoptotic genes promoters. The variability of pathway activation (i.e. p53-CC, apoptosis vs wt-p53, cell cycle arrest) may be specific to this tumor model due to the dominant negative mutant p53 endogenously found in MDA-MB-468 cells/tumors. This is because we have shown previously 12 that p53-CC and wt-p53 induce similar levels of apoptosis in four different non-p53-dominant negative breast cancer cell lines with varying endogenous p53 statuses (H1373 cells: p53 null, HeLa cells: wt-p53, T47D cells: wt-p53 mislocalized, and MDA-MB-231 cells: mutant p53). 12 Furthermore, qRT-PCR analyses and western blotting showed that p53-CC and wt-p53 induced similar levels of p21 gene expression in T47D breast carcinoma cells.
In summary, we have shown for the first time, use of a version of p53 that overcomes the limitations of using wt-p53 for gene therapy. A chimeric superactive p53 has been described as the 'ultimate cancer therapeutic'. 44 Our p53-CC demonstrates comparable functional transcriptional activity to wt p53, 12 shows significantly improved apoptosis (Figures 2 and  3), and successfully circumvents the dominant negative inactivating effect of endogenous mutant p53 in vitro. 12 Importantly, our compelling in vivo data (Figure 4) demonstrates that p53-CC is more effective than wt p53, and may serve as a more potent and reliable novel anticancer therapeutic. Okal

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Recombinant Adenovirus Production
Replication-deficient recombinant adenovirus serotype 5 (Ad) constructs were generated using the Adeno-X ® Adenoviral Expression System 3 (Clontech, Mountain View, CA) As we have done before. 12 Either wt-p53 or p53-CC was inserted into a cassette under the control of the CMV promoter. A separate CMV promoter controls the expression of ZsGreen1 fluorescent protein for visualization. The empty virus (vector) was used as a negative control. Wt-p53 and p53-CC were PCR amplified with primers containing 15 base pair homology with a linearized pAdenoX vector (Clontech) based on an In-Fusion ® HD Cloning Kit (Clontech). Stellar ® competent cells (Clontech) were transformed with the adenoviral vector plasmids containing our constructs. Viral DNA was then purified, linearized and transfected into HEK293 cells for packaging and amplification. Viral particles were isolated from HEK293 cells by freeze-thawing, purified using Adeno-X ® Mega Purification Kit (Clontech), and dialyzed against storage and proper tonicity buffer (2.5% glycerol (w/v), 25 mM NaCl, and 20 mM Tris-HCl, pH 7.4). The viral titer was determined using flow cytometry per the manufacturer's recommendation.

Cell Lines and Viral Transductions
HEK293 human embryonic kidney cells (ATCC, Manassas, VA) were used for viral production and MDA-MB-468 human breast adenocarcinoma cells (ATCC) harboring a dominant negative mutant p53 were grown as monolayers in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-glutamine, and 0.1% gentamicin. MDA-MB-468 cells were also supplemented with 1% MEM non-essential amino acids (Invitrogen). All cells were incubated in 5% CO 2 at 37°C. The cells were seeded at a density of 3.0 × 10 5 cells in 6-well plates (Greiner Bio-One, Monroe, NC). Viral transductions were carried out immediately after seeding the cells at multiplicity of infection (MOI) of 200.

7-AAD Assay
Following manufacturer's instructions and as previously described, 45 MDA-MB-468 cells were stained with 7-aminoactinomycin D (7-AAD, Invitrogen) 48 h after transfection. Cells were analyzed and gated for ZsGreen1 (with same fluorescence intensity to ensure equal expression of proteins) using the FACSCanto-II (BD-BioSciences, University of Utah Core Facility) and FACSDiva software. Excitation was set at 488 nm and detected at 507 nm and 780 nm for ZsGreen1 and 7-AAD, respectively. The means from three separate experiments (n=3) were analyzed using one-way ANOVA with Bonferroni's post hoc test.

TMRE Assay
MDA-MB-468 cells were incubated with 100 nM tetramethylrhodamine ethyl ester (TMRE) (Invitrogen) for 30 min at 37 °C 36 h after infection. 46 The time point was determined to be 36 h as a result of several optimization pilot studies for the TMRE assay, and since mitochondrial outer membrane permeabilization occurs prior to caspase-3/7, annexin-V, and 7-AAD detection (48 h). MDA-MB-468 cells were pelleted and resuspended in 300 µL of annexin-V binding buffer (Invitrogen). Only ZsGreen1 positive cells were analyzed by using the FACS Canto-II (BD BioSciences, University of Utah Core Facility) with FACS Diva software. ZsGreen1 was excited with the 488 nm laser with emission filter 530/35, and TMRE was excited with the 561 nm laser with the emission filter 585/15. Mitochondrial depolarization (loss in TMRE intensity) correlates with an increase in MOMP. 18 Independent transfections of each construct were tested three times (n = 3).

Annexin-V Assay
The annexin-V assay was performed as before. 12,45 Briefly, 48 h post infection, MDA-MB-468 cells were suspended in 400 µl annexin binding buffer (Invitrogen) and incubated with 5 µl annexin-APC (annexin-V conjugated to allophycocyanin, Invitrogen) for 15 minutes. The incubated cells were ZsGreen1 gated and analyzed using FACSCanto-II. ZsGreen1 and APC were excited at 488 nm and 635 nm wavelengths and detected at 507 nm and 660 nm, respectively. Each construct was tested three times (n=3) and analyzed using one-way ANOVA with Bonferroni's post hoc test. 45

In Vivo Study
The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah. All experiments were performed in Female nu/nu athymic mice (6-8 weeks old, Jackson Laboratories, Bar Harbor, ME). Human MDA-MB-468 cells (1×10 7 cells/mouse in 100 µl of serum-free RPMI-1640 medium) were injected subcutaneously into the mammary fat pad located in the right inguinal area. When tumors reached a mean size of 50mm 3 , animals were randomized into 4 treatment groups and received single peritumoral injections of adenoviral constructs (5.0×10 8 pfu) in a 50 µl volume prepared fresh on days 0-4 and 7-11. Twenty four hours after the last injection the mice were sacrificed and the tumors as well as the organs were harvested for analyses. Tumor volumes were measured daily using Vernier calipers along the longest width (W) and the corresponding perpendicular length (L). The tumor volume was calculated using V= (L × W (0.5W)). All procedures were performed according to established NIH guidlines and University of Utah Institutional Animal Care and Use Committee approved protocols.

Histology
Animal tumor tissue samples and organs were fixed in 10% formalin for 24 h followed by tissue preparation and embedded in paraffin. Embedded tissues were then sectioned to cut at 4 µm thick sections and mounted on plus slides. Slides from each tumor tissue from all mice in the three treatment groups as well as the untreated group were stained using hematoxylin and eosin and p21 immunohistochemistry stain. Tissue and histological slide preparation was conducted in collaboration with ARUP Laboratories (Salt Lake City, Utah). 47
In vitro: 24 h following infection of MDA-MB-468 cells, 3×10 5 cells were pelleted and resuspended in 200 µL lysis buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 1 % protease inhibitor), sonicated on ice, and centrifuged for 15 min at 14,000 rpm. The supernatants were used for immunoblotting as described above and densitometry analysis was performed as described before. 49

Cell Cycle Arrest
The cell cycle arrest analysis was carried out as previously described. 48 Briefly, 1 × 10 6 MDA-MB-468 cells were infected as described above (Cell Lines and Viral Transductions

Statistical Analysis
One-way ANOVA with Bonferroni's post test was used to compare the different treatment groups and controls. A value of p < 0.05 was considered statistically significant. Error bars represent standard deviations from at least three independent experiments (n = 3) except for the animal study (n=6).       Gene Ther. Author manuscript; available in PMC 2015 April 01.