Susceptibility of cancer cells to herpes simplex virus-dependent apoptosis

Apoptosis is initially triggered early in a herpes simplex virus 1 (HSV-1) infection through the transcription of a specific immediate-early viral gene, ICP0 (Sanfilippo & Blaho, 2006; Sanfilippo et al., 2004). During the later stages of the virus life cycle, anti-apoptotic proteins are synthesized, which prevent apoptotic cell death from proceeding. This produces a delicate balance of apoptotic signals during infection that allows the cells to produce progeny virions prior to apoptotic cell death (reviewed by Nguyen & Blaho, 2007). However, in situations where the preventors are not produced efficiently, the balance is upset and the infected cells die by apoptosis. To date, at least one cellular (Goodkin et al., 2003; Gregory et al., 2004; Yedowitz & Blaho, 2005) and seven viral survival factors (Aubert & Blaho, 1999; Aubert et al., 1999; Gupta et al., 2006; Jerome et al., 1999, 2001; Leopardi & Roizman, 1996; Leopardi et al., 1997; Perng et al., 2000) have been identified and implicated in the apoptotic prevention process. One essential viral preventer of apoptosis is the immediate-early ICP27 protein. ICP27-null viruses are able to trigger but not prevent apoptotic cell death, and cells infected with these viruses die (Aubert & Blaho, 1999; Aubert et al., 2001) through a process that we refer to here as HSV-1-dependent apoptosis (HDAP).

Much of the initial characterization of HDAP utilized the HEp-2 strain of HeLa cells (Chen, 1988; Nelson-Rees et al., 1974; Ogura et al., 1993) and these studies provided information crucial for understanding the viral factors involved in modulating the process (reviewed by Aubert & Blaho, 2001). When studies were expanded to include other cell types, it became apparent that a range of sensitivities to HDAP exists. For instance, Vero cells, which are a primate kidney cell line typically used for propagating HSV-1, did not exhibit HDAP at early time points (Aubert & Blaho, 1999). Further studies revealed that Vero cells do undergo HDAP, albeit at later times post-infection than HeLa cells (Nguyen et al., 2005). The inhibition of protein synthesis during infection reduced HDAP in Vero cells, demonstrating that proteins newly synthesized early in infection facilitate HDAP. Synthesis of these proteins is essential for efficient HDAP in Vero but not HeLa cells, highlighting a fundamental difference in the way these two cell lines respond to this process. Although both HeLa and Vero cells have an indefinite life span (i.e. they are immortalized), only HeLa cells display the anchorage-independent growth needed to form a tumour (i.e. they are transformed) (Contreras et al., 1985). In contrast to both HEp-2 and Vero cells, primary murine and human fibroblast cells were completely resistant to apoptosis induced by HSV-1 (Aubert & Blaho, 2003).

In this study, we set out to address whether transformation status could explain the differences in the response to HDAP. The susceptibility of human cancer cells derived from various types of tumour was assessed. Cells derived from normal tissue, peripheral to a mammary tumour, were resistant to HDAP, whilst the syngeneic cancer cells were susceptible, indicating that genetic lesions occurring during tumorigenesis sensitized these cells. The susceptibility of cells derived from colon, brain, breast and cervical cancers to HDAP was determined. Two cell lines were resistant to HDAP, but they were also highly resistant to exogenous apoptotic stimuli. These resistant cells have probably acquired additional mutations that target their cellular apoptotic machinery. Together, these results indicate that the efficiency of the cellular apoptotic response is a determinant that is capable of altering susceptibility to HDAP.

Cells and viruses.
All cells were obtained from ATCC. U373, SK-N-SH, RKO and RKO-E6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS). Hs578T cells were grown in DMEM containing 10 % FBS and 0.01 mg bovine insulin ml¹. HT-29 and PC-3 cells were grown in 10 % FBS-containing McCoys 5a or F12K medium, respectively. Hs578Bst cells were grown in Hybri-Care (ATCC) medium supplemented with 10 % FBS and 30 ng epidermal growth factor ml¹. Hs578Bst cells are a diploid cell line that we obtained from ATCC at passage 9. In preliminary experiments, we observed that these cells enter a non-growing state around passage 16, similar to that observed for primary epithelial cells. Therefore, all experiments were performed using Hs578Bst cells used at passages prior to passage 14. Human mammary epithelial cells (HMECs) were grown in mammary epithelial growth medium (both from Cambrex). The HMECs used in this study had undergone approximately four population doublings since purchase. Vero 2.2 and HEp-2 cells were maintained in DMEM with 5 % FBS. Vero 2.2 cells (a gift from Saul Silverstein, Columbia University, NY, USA) are derivatives of Vero cells expressing ICP27 from its viral promoter (Sekulovich et al., 1988). HSV-1 KOS1.1 was the strain of wild-type HSV-1 used in this study. HSV-1 strain vBSΔ27 is an ICP27-null virus derived from HSV-1 KOS 1.1 containing a replacement of the α27 gene with the Escherichia coli lacZ gene (Soliman et al., 1997). This virus was propagated and titrated on Vero 2.2 cells and used to infect cells at an m.o.i. of 10, as reported previously (Nguyen et al., 2005). CgalΔ3 was derived from HSV-1 strain 17syn⁺ and is an IE3 (ICP4)-null virus that has a deletion of 3.6 kb of the coding region of IE3 (Paterson et al., 1990) due to insertion of the Escherichia coli lacZ gene in the BamHI Z fragment (Johnson et al., 1992). As described previously (Nguyen et al., 2005), CgalΔ3 was propagated and titrated on FO6 cells, which are derived from Vero cells and express ICP27, ICP4 and ICP0 from their own promoters (Samaniego et al., 1997). In experiments designed to inhibit protein synthesis, cycloheximide (CHX) was added directly to the medium at a concentration of 10 µg ml¹ 1 h prior to infection and maintained at that level until the time of harvest. As we observed previously (Aubert & Blaho, 1999), it was expected that certain cells, particularly highly transformed lines, may have increased resistance to CHX. With this caveat, we chose the concentration of CHX that is our standard amount required for complete inhibition of transformed human HEp-2 cells. Tumour necrosis factor (TNF; 10 ng ml¹) plus CHX (10 µg ml¹) or staurosporine (STS; 1 µM) were added to cells as a positive control for apoptosis induction. Unless otherwise noted, all cell-culture reagents were obtained from Life Technologies and all biochemicals from Sigma.

Microscopic analysis and monitoring of chromatin condensation.
The morphology of infected cells was documented by phase-contrast and fluorescence microscopy using an Olympus IX70/IX-FLA inverted fluorescence microscope. Images were acquired using a Sony DKC-5000 digital photo camera linked to a PowerMac workstation and processed through Adobe Photoshop. For visualization of chromatin condensation in live cells, 5 µg Hoechst 33258 (Sigma) ml¹ was added to the medium and allowed to incubate at 37 °C for 30 min. The percentage of nuclei containing condensed chromatin was determined by dividing the number of brightly stained, small (condensed) nuclei by the total number of nuclei (uncondensed plus condensed) in a particular (x40) microscopic field. At least 100 nuclei were counted for each data point. For Fig. 1, the percentage of chromatin condensation is represented as the mean±SD of three independent experiments.

(80K): Fig. 1. HSV-1-dependent apoptosis occurs in mammary tumour cells, but not in syngeneic normal breast cells. (a) Nuclear (Hoechst) and cellular (phase) morphologies. Hs578T tumour and Hs578Bst normal cells were visualized at 24 h following infection with wild-type HSV-1 (KOS) or apoptotic vBSΔ27 (Δ27) (m.o.i.=10) or at 24 h post-treatment with STS. Magnification, x40. Insets inside Hoechst panels represent enlarged images of nuclei from each panel to aid in visualization. The values in the lower right corner of the Hoechst panels denote the mean±SD of the percentage of nuclei containing condensed chromatin from three independent experiments assessed as described in Methods. (b, c) Immunoblots of death factors (PARP, DFF-45 and procaspase 3) (b) and viral proteins (ICP4, ICP27, TK and gC) (c) in Hs578T and Hs578Bst cells at 24 h post-treatment with STS or post-infection with HSV-1 (KOS) or vBSΔ27 (Δ27) in the presence (+) or absence () of CHX. The positions of the 116 kDa uncleaved and 85 kDa cleaved PARP products are indicated in the right-hand margin. Some slight spreading of the bottom of the gel occurred in the very low molecular mass range such that the panel showing procaspase 3 does not align with the other panels.

Immunoblotting.
Whole-cell protein extract was prepared using lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 % deoxycholate, 0.1 % SDS) supplemented with 2 mM PMSF (freshly prepared stock), 1 % Translysol, 0.1 mM L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone, 0.01 mM L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride, as previously reported (Nguyen et al., 2005). Protein concentrations were determined using a modified Bradford protein assay (Bio-Rad Laboratories). Total protein (20 or 50 µg) was separated on 15 % N,N'-diallyltartardiamide-acrylamide gels and electrically transferred to nitrocellulose. Pre-stained molecular mass markers were loaded (not shown) and immunostaining of the actin loading control was carried out. Membranes were incubated for 1 h at room temperature in blocking buffer (PBS containing 5 % non-fat, dried milk) and incubated overnight at 4 °C in primary antibody. Monoclonal antibodies specific for ICP4, ICP27, gC (all from the Goodwin Institute for Cancer Research), poly(ADP-ribose) polymerase (PARP) (PharMingen), procaspase 3 (BD Transduction) and the control actin (Sigma) and polyclonal antibodies specific for thymidine kinase (TK) and DFF-45 (Santa Cruz) were diluted at a concentration of 1 : 1000 in Tris-buffered saline containing 0.1 % Tween 20 (TBST) and 0.1 % BSA. After washing in TBST, membranes were incubated with either anti-mouse or anti-rabbit antibodies conjugated to alkaline phosphatase (Southern Biotech) diluted in blocking buffer (1 : 1000) for 1 h at room temperature. Following washing in TBST, immunoblots were developed in buffer containing 5-bromo-4-chloro-3-indolyl phosphate and 4-nitro blue tetrazolium chloride.

Densitometric analysis.
To quantitate the percentage of total infected cell PARP that was cleaved, densitometry of immune-reactive PARP was performed as described previously (Aubert et al., 1999). NIH IMAGE version 1.63 was used to measure the integrated density (ID) of the 116 kDa uncleaved and 85 kDa cleaved PARP bands. These values were used to calculate the percentage of PARP cleavage for each lane using the following formula: % cleavage=[(cleaved PARP ID)/(cleaved PARP ID plus uncleaved PARP ID)]x100 %.

An intricate balance between pro- and anti-apoptotic processes exists in HSV-1-infected cells, which enables productive virus propagation. When anti-apoptotic pathways are suppressed during infection, this balance is upset and the cells die by apoptosis. In previous studies (Aubert & Blaho, 2003; Nguyen et al., 2005), we observed that HeLa cancer cells exhibited an enhanced sensitivity to HDAP, which implicated oncogenic pathways in HDAP regulation. The goal of this study was to utilize a series of cancer cells to determine whether their transformation status was sufficient to be a determinant of apoptotic killing by HSV-1.

HSV-1-dependent apoptosis occurs in mammary tumour cells, but not syngeneic normal breast cells
Whilst earlier studies provided evidence that the sensitivity to HDAP is linked to transformation status (Aubert & Blaho, 2003; Nguyen et al., 2005), a direct comparison of syngeneic tumour and normal cells was not done. Our first experiment compared Hs578T mammary tumour cells with normal epithelial (Hs578Bst) cells derived from tissue peripheral to the tumour (Hackett et al., 1977). All experiments with the Hs578Bst cells were performed using cells with passage numbers less than 14. To assess the ability of these cells to undergo HDAP, Hs578T and Hs578Bst cells were mock infected or infected with wild-type HSV-1 strain KOS1.1 (KOS) or with an ICP27-null recombinant virus, vBSΔ27. In addition, as we have recently determined that at least one cell line requires de novo protein synthesis to undergo HDAP (Nguyen et al., 2005), we also assessed the role of protein synthesis by performing the experiments in the presence and absence of the protein synthesis inhibitor CHX. STS treatment was also used as a positive control for apoptosis induction. Apoptosis was evaluated at 24 h post-treatment by monitoring morphological changes, chromatin condensation, procaspase 3 and DFF-45 protein levels and cleavage of the caspase 3 substrate, PARP, from its 116 kDa form into an 85 kDa fragment.

Mock-infected Hs578T and Hs578Bst cells were flat and well spread out and their nuclei exhibited homogeneous Hoechst staining (Fig. 1a). In contrast, KOS-infected Hs578T cells exhibited an enlarged, rounded morphology, and bright Hoechst staining was evident in the periphery of their nuclei. These morphological changes are characteristic of the cytopathic effect (CPE) that accompanies productive HSV-1 replication (Avitabile et al., 1995; Hampar & Elison, 1961; Heeg et al., 1986; Roizman, 1962, Roizman & Roanne, 1964). Although the CPE in Hs578Bst cells was more subtle than that of KOS-infected Hs578T cells, their morphology differed from that of mock-infected cells. Specifically, the nuclei were larger, with brighter Hoechst staining around the periphery compared with mock-infected cells, which had uniform staining. In addition, a prominent ridge was evident around the nuclei in the light microscopy images of KOS-infected Hs578Bst cells, which is a common feature of cells undergoing productive HSV-1 infection. The KOS-infected cells of both cell types produced similar levels of representative immediate-early (ICP4 and ICP27), early (TK) and late (gC) viral proteins (Fig. 1c, lanes 3 and 9). These results indicated that the Hs578T and Hs578Bst cells were capable of supporting HSV-1 infection with similar efficiencies. STS-treated Hs578T and Hs578Bst cells were smaller and irregular shaped compared with the mock-treated cells (Fig. 1a). In addition, they exhibited membrane protrusions characteristic of membrane blebbing. The nuclei of STS-treated cells were smaller in size than those of mock-treated cells and contained regions of intense Hoechst staining indicative of chromatin condensation. When this phenotype was quantified for three independent experiments, the Hs578T and Hs578Bst cells exhibited 86±14 % and 74±44 % chromatin condensation, respectively. The lysates of STS-treated Hs578T and Hs578Bst cells also displayed a band corresponding to the cleaved 85 kDa product of PARP, and procaspase 3 and DFF-45 protein levels were drastically reduced from that of mock-infected cells (Fig. 1b, compare lane 7 with 1 and lane 14 with 8). As the STS-treated Hs578T and Hs578Bst cells exhibited the morphological and biochemical characteristics of apoptosis, we concluded that both cell types were capable of undergoing apoptosis. Thus, the primary Hs578Bst cells were not senescent and not generally resistant to apoptosis.

Fifty-three per cent of the Hs578T cells infected with vBSΔ27 exhibited membrane blebbing and chromatin condensation (Fig. 1a). Additionally, they displayed PARP cleavage and had lower levels of DFF-45 and procaspase 3 (Fig. 1b, lane 5) than mock-infected cells (Fig. 1b, lane 1). These results demonstrated that the Hs578T cells were sensitive to HDAP. Similarly, infection with KOS or vBSΔ27 in the presence of CHX led to apoptotic morphologies (data not shown) and reductions in DFF-45 and procaspase 3 (Fig. 1b, lanes 4 and 6) in these cells. Although CHX treatment led to some background PARP cleavage in mock-infected Hs578T cells, significantly more PARP cleavage was evident in cells treated with KOS plus CHX and vBSΔ27 plus CHX (Fig. 1b, compare lane 2 with lanes 4 and 6). Importantly, significant PARP and complete DFF and procaspase 3 processing was observed with vBSΔ27-infected cells in the absence of CHX, indicating that this was not simply due to CHX. Together, these results demonstrated that Hs578T cells underwent HDAP in a manner similar to the HEp-2 cells and that they did not require de novo protein synthesis for this process to occur.

In contrast, only a very small percentage (4±4 %) of the vBSΔ27-infected Hs578Bst cells exhibited chromatin condensation (Fig. 1a). This level was comparable to that seen in KOS-infected Hs578Bst cells (5±9 %). Furthermore, neither detectable PARP cleavage nor reductions in DFF-45 or procaspase 3 were observed in Hs578Bst cells that were infected with vBSΔ27 (Fig. 1b, compare lanes 12 and 8). This result indicated that, although the Hs578Bst cells were sensitive to STS-induced apoptosis, they were resistant to HDAP. Hs578Bst cells treated with KOS or vBSΔ27 plus CHX also failed to undergo apoptosis (Fig. 1b, compare lanes 11 and 13 with lane 8). Together, the data presented in Fig. 1 demonstrated that the normal tissue-derived Hs578Bst cells were resistant to HDAP, whilst the tumour-derived Hs578T cells were sensitive.

Primary HMECs are resistant to HDAP
The differential sensitivity of primary Hs578Bst and transformed Hs578T cells suggested that genetic changes occurring during tumorigenesis sensitized the tumour cells to HDAP. This could reflect a general sensitivity of tumour cells to pro-apoptotic stimuli, which is the basis of certain chemotherapy treatments. However, the primary Hs578Bst cells were originally derived from normal tissue and the aliquot that we received from ATCC was from passage number 9. Therefore, it was possible that these cells had acquired genetic mutations during their subculturing that had rendered them resistant to HDAP. Thus, we tested the susceptibility of low-passage-number primary normal HMECs to HDAP. To accomplish this, primary HMECs (Cambrex) grown in defined growth medium were infected with wild-type KOS and vBSΔ27 at an m.o.i. of 10. Twenty-four hours later, the cells were assessed for chromatin condensation, PARP cleavage and the presence of viral proteins. Cells infected with KOS exhibited CPE (Fig. 2a) and expressed the ICP4, gC and ICP27 viral proteins (Fig. 2b, lane 2). As expected, ICP4, but not ICP27 or gC, was detected in the lysate of vBSΔ27-infected cells (Fig. 2b, lane 3). These results confirmed that the primary HMECs were infected efficiently with KOS and vBSΔ27. However, the vBSΔ27-infected primary HMECs did not display chromatin condensation (Fig. 2a). Furthermore, we did not detect any PARP cleavage or reductions in DFF-45 and procaspase 3 protein levels with vBSΔ27 (Fig. 2b, lane 3). We observed a similar apoptotic resistance of separate isolations of primary HMECs (data not shown). We consistently observed increases in the amounts of procaspase 3 and DFF relative to mock- and KOS-infected cells during vBSΔ27 infection (Fig. 2b, lane 3). The basis of this is unknown, but it further confirmed the lack of apoptosis in these cells. From these findings, we concluded that primary HMECs are resistant to HDAP. These results, along with those in Fig. 1, are significant as they represent the first characterization of primary human epithelial cells infected with an HSV strain that results in apoptosis of at least two (HeLa and Hs578T) types of cancer cell.

(91K): Fig. 2. Primary HMECs are resistant to HSV-1-dependent apoptosis. (a) Nuclear (Hoechst) and cellular (phase) morphologies. Primary HMECs were visualized at 24 h following HSV-1 (KOS) and vBSΔ27 (Δ27) infection (m.o.i.=10). The values in the lower-right corner of the Hoechst panels denote the mean±SD of the percentage of nuclei containing condensed chromatin, assessed as described in Methods. Magnification, x40. (b) Immunoblots of death factors (PARP, procaspase 3, DFF-45), viral proteins (ICP4, ICP27, gC) and the control actin at 24 h post-infection. The positions of the 116 kDa uncleaved and 85 kDa cleaved PARP products are indicated in the right-hand margin.

The susceptibility of cancer cells to HSV-1-dependent apoptosis correlates with sensitivity to exogenous environmental apoptotic inducers
The results described for Fig. 1 demonstrated that at least one breast cancer cell line is sensitive to HDAP. Previously published studies have shown that HeLa/HEp-2 cancer and 143 tumour cell lines, typically used to study HSV-1 replication, are sensitive to HDAP (Aubert & Blaho, 1999, 2003; Koyama & Adachi, 1997). However, transformed human embryonic kidney 293 cells and caspase 3-null MCF-7 breast cancer cells are resistant to HDAP (Aubert & Blaho, 2003; Kraft et al., 2006), indicating that not all tumour cells are equally susceptible to HDAP. To gain further insight into the cancer-cell determinants for susceptibility to HDAP, we analysed a broader range of tumour cells. Specifically, we assessed the sensitivity of cell lines derived from colon (HT-29, RKO and RKO-E6), prostate (PC-3) and brain (SK-N-SH and U373) tumours to HDAP. HEp-2 cells were used as a control cell line that was known to be sensitive to HDAP (Aubert & Blaho, 2003).

Each cell line was infected with KOS, vBSΔ27 and/or another recombinant virus that lacked expression of ICP4, CgalΔ3. Like vBSΔ27, CgalΔ3 triggers but does not prevent apoptosis during infection (Aubert & Blaho, 2003). We also assessed the role of protein synthesis during HDAP treatment in these cell lines by adding CHX to a subset of the infections. STS and/or TNF plus CHX were used as positive controls for apoptosis. At 24 h p.i., chromatin condensation was monitored via Hoechst staining. Subsequently, cells were harvested and immunoblotted for the accumulation of viral proteins and biochemical markers of apoptosis. Cell morphologies and immunoblot results from the HT-29, RKO and SK-N-SH cells are presented in Figs 3 and 4. The results from PC-3 and U373 cells are displayed in Fig. 5. The HEp-2 cells are presented in each figure for comparison.

(96K): Fig. 3. Cancer cells sensitive to exogenous apoptotic stimuli are sensitive to HSV-1-dependent apoptosis. Cellular (phase) and nuclear (Hoechst) morphologies of HEp-2, HT-29, SK-N-SH and RKO cells at 24 h following infection with wild-type HSV-1 (KOS) or vBSΔ27 (Δ27) at an m.o.i. of 10 or treatment with STS. Magnification, x40. The values in the lower right-hand corner of the Hoechst panels denote the percentage of nuclei containing condensed chromatin assessed as described in Methods.

(50K): Fig. 4. Cancer cells sensitive to exogenous apoptotic stimuli are sensitive to HSV-1-dependent apoptosis. Immunoblots of death factors (PARP and procaspase 3) and viral proteins (gC, ICP27, ICP4 and VP22) from HEp-2 (a), HT-29 (b) RKO (c) and SK-N-SH (d) cells at 24 h post-treatment with STS or TNF or infection with HSV-1 (KOS), vBSΔ27 (Δ27) or CgalΔ3 (Δ3) at an m.o.i. of 10 in the presence (+) or absence () of CHX. The positions of the 116 kDa uncleaved and 85 kDa cleaved PARP products are indicated in the right-hand margin. The percentage of PARP cleavage (%Cleav) was calculated as described in Methods.

(86K): Fig. 5. Cancer cells that are resistant to exogenous apoptotic stimuli are resistant to HSV-1-dependent apoptosis. (a) Cellular morphologies of HEp-2, PC-3 and U373 cells were visualized at 24 h following HSV-1 (KOS) and vBSΔ27 (Δ27) infection (m.o.i.=10) or 24 h post-treatment with STS. Magnification, x40. The values in the lower-right corner of the Hoechst panels denote the percentage of nuclei containing condensed chromatin assessed as described in Methods. (b)(d) Immunoblots of death factors (PARP and procaspase 3) and viral proteins (ICP4, ICP27 and gC) from HEp-2 (b), PC-3 (c) and U373 (d) cells at 24 h post-treatment with STS or infection with HSV-1 (KOS) or vBSΔ27 (Δ27) in the presence (+) or absence () of CHX. The positions of the 116 kDa uncleaved and 85 kDa cleaved PARP products are indicated in the right-hand margin. The percentage of PARP cleavage (%Cleav) was calculated as described in Methods.

All of the cell lines tested displayed CPE (Figs 3 and 5a) and had similar levels of accumulation of the ICP27 viral protein following KOS infection (Fig. 4 and Fig. 5bd, lane 3), indicating that they were equally susceptible to viral infection. STS treatment led to abundant apoptotic morphologies in the HT-29, RKO and SK-N-SH cell lines (Fig. 3). Additionally, 50 % of RKO and 30 % of HT-29 nuclei exhibited chromatin condensation following STS treatment. It was not possible to assess chromatin condensation in SK-N-SH cells in this manner due to a high level of background Hoechst staining in these live cells (data not shown). STS induced almost complete (>95 %) PARP cleavage in HT-29, RKO and SK-N-SH cells, as well as reductions in procaspase 3 levels (Fig. 4b and d, lane 7; Fig. 4c, lane 9). Together, these results indicated that these cell lines were sensitive to STS-induced apoptosis. TNF plus CHX treatment similarly induced apoptotic morphologies (data not shown) and death factor processing in these cells (Fig. 4b, lane 8, and data not shown).

Like HEp-2 cells, which are susceptible to HDAP, vBSΔ27-infected HT-29, RKO and SK-N-SH cells exhibited membrane blebbing (Fig. 3). Infection with vBSΔ27 led to 50 and 23 % of nuclei with condensed chromatin in HT-29 and RKO cells, respectively. The lysates from all of these cell lines also displayed PARP cleavage levels of between 51 and 60 % and small reductions in procaspase 3 when infected with vBSΔ27 (Fig. 4b, c, lane 5). In addition, RKO cells infected with CgalΔ3 exhibited 37 % PARP cleavage (Fig. 4c, lane 7), indicating that these cells are susceptible to HDAP induced by multiple recombinant viruses. We observed that infection with KOS led to 94 % PARP cleavage in the SK-N-SH cells. Together, these results demonstrated that HT-29, RKO and SK-N-SH cell lines are sensitive to HDAP. Other investigators using different assays have observed low levels of apoptosis in wild-type HSV-infected SK-N-SH cells (Galvan & Roizman, 1998; Peng et al., 2005). It should be noted that our SK-N-SH cells were used directly from ATCC and were at low passage (<20). Therefore, our results seemed to indicate that the SK-N-SH cells are unable to set up a perfect apoptotic balance, even in the presence of viral apoptotic preventors. This may be due to a heightened sensitivity for apoptosis in these cells, as even mock-infection led to a relatively high level of PARP cleavage (29 %, Fig. 4d, lane 1).

RKO, HT-29 and SK-N-SH cells also demonstrated apoptosis when infected with vBSΔ27 or KOS in the presence of CHX (data not shown and Fig. 4bd, lanes 4 and 6), consistent with HDAP occurring independently of de novo protein synthesis in these cells. RKO cells expressing the human papillomavirus E6 protein (RKO-E6) exhibited an identical response to RKO cells with respect to both STS and HDAP (data not shown). From these results, we concluded that certain colon and brain tumour-derived cells can respond to HDAP.

In contrast, PC-3 and U373 cells did not exhibit substantial apoptotic morphology (Fig. 5a), PARP cleavage, or a reduction in procaspase 3 when treated with STS (Fig. 5c, d, lane 7) or TNF plus CHX (Fig. 5d, lane 8, and data not shown), indicating that these cell lines are more resistant than the aforementioned cell lines. Strikingly, the PC-3 and U373 cells displayed little to no membrane blebbing following infection with vBSΔ27. Only 5 % of the vBSΔ27-infected PC-3 cells displayed chromatin condensation (Fig. 5a). Furthermore, PARP was found only in the uncleaved form and procaspase 3 levels did not change following vBSΔ27 infection (Fig. 5c, d). This result indicated that PC-3 and U373 cells are resistant to HDAP. KOS and vBSΔ27 infections in the presence of CHX also failed to cause apoptosis in these cells. Together, the results from Figs 35 demonstrated a correlation between the sensitivity to HDAP and the response to environmental apoptotic stimuli in cancer cells.

HSV modulates apoptosis during the course of its productive infection (reviewed by Nguyen & Blaho, 2007) and this probably plays an important role in the pathogenesis of herpesviral disease (Miles et al., 2003; Sabri et al., 2006). However, recent studies have suggested that there is an unexpected pattern of cell susceptibility to HDAP, as tumour cells appear to be exquisitely sensitive to this cell-death process (Aubert & Blaho, 2003). Accordingly, the goal of this study was to assess whether cell transformation status was the sole determinant of HDAP. Our key findings may be summarized as follows.

Sensitivity to environmentally induced apoptosis predicts cancer cell susceptibility to HDAP. We now have a large body of information on the response of numerous cell types to HDAP (Table 1). The striking finding is that cells that are sensitive to apoptotic cell death triggered by exogenous agents are also able to be killed by HDAP. All of these HDAP-susceptible cells were treated with and found to be sensitive to the intrinsic inducer STS. Of these cells that were also treated with TNF plus CHX, this group was also sensitive to this extrinsic method of induction. Thus, these cells possess the necessary internal apoptotic machinery to respond to all types of pro-apoptotic stimuli. It has recently been shown that HDAP occurs as a result of cytochrome c release from mitochondria, which occurs independently of caspase activation, and, thus, implicates the intrinsic apoptotic pathway as the response to virus (Aubert et al., 2007). Due to the implicit cross-talk that occurs from the extrinsic to the intrinsic pathways (reviewed by Sanfilippo & Blaho, 2003), we must conclude that, in order for a cancer cell to be susceptible to HDAP, it must possess the intact cellular machinery of the mitochondrial-dependent apoptotic cascade. Our findings should be of interest to those studying virus-induced apoptosis and the virotherapy of cancer. We have already shown that viruses singly deleted for either the HSV ICP4 (this study, and Aubert & Blaho, 2003; Nguyen et al., 2005) or ICP22 (Aubert et al., 1999; Sanfilippo & Blaho, 2006) regulatory protein also possess the ability to induce HDAP in certain human tumour cells. It is conceivable that other viruses possessing deletions in certain accessory apoptosis prevention factors (reviewed by Aubert & Blaho, 2001; Goodkin et al., 2004), such as U_S3 (Jerome et al., 1999; Leopardi et al., 1997), might have some level of HDAP efficacy.

Table 1. Cancer cell susceptibilities to HDAP correlate with sensitivities to environmentally induced apoptosis

Not all cancer or tumour cells die by HDAP. In contrast, those cancer cells that exhibited resistance to HDAP seemed to possess a general apoptotic defect (Table 1). Of course, this conclusion carries at least one major caveat. Whilst we cannot rule out the possibility that PC-3 and U373 cells can undergo apoptosis when exposed for longer time periods or with higher doses of apoptotic inducers, this may not be significant if the time that it takes to detect markers of apoptosis exceeds the virus replication cycle. Nevertheless, it was clear that such cells were more resistant to STS and TNF plus CHX treatments than the cells that were sensitive to HDAP. Although initial oncogene activation or tumour suppressor inhibition commonly renders certain cells more susceptible to apoptosis, it has been postulated that subsequent mutations selected for during tumorigenesis render these cells less sensitive (reviewed by Brown & Wouters, 1999). Importantly, these mutations often affect the functions of proteins central to apoptosis execution. In fact, PC-3 cells have been reported to overexpress the anti-apoptotic Bcl-2 family member Bcl-X_L (Liu & Stein, 1997), which is probably the reason they failed to display HDAP. It is conceivable that the U373 cells contain similar genetic alterations that render them highly resistant to apoptosis. Further support for this mutational defect model comes from our recent findings that the caspase 3-null breast cancer cells MCF-7 are resistant to HDAP (Table 1). However, MCF-7 cells that were reconstituted for caspase 3 (MCF-7/C3) underwent HDAP (Kraft et al., 2006). In the case of the laboratory-transformed HEK 293 cells, the basis of the apoptotic resistance is the integrated (Graham et al., 1977) presence of adenovirus anti-apoptotic genes (reviewed by Lichtenstein et al., 2004). In summary, it is likely that mutations in the cellular apoptotic machinery are responsible for the cancer cell resistance to HDAP found in this study.

All primary cells tested were resistant to HDAP. The fact that a syngeneic pair of tumour and normal cells exhibited opposite responses to HDAP treatment strongly argues that alterations in cancer-related genes are responsible for the tumour-specific cell death. The inability of primary cells to die by HDAP seemed to be specific to the virus as they were all still sensitive to other environmental apoptotic inducers including STS and TNF plus CHX (Table 1). The fact that these cells were able to die by exogenous apoptosis induction indicated that they were not senescent and did not possess a general resistance.

The consistently reproducible inability of primary cells to die by HDAP represents one of the most intriguing and complicated facets of the analysis of apoptosis during HSV infection. Recognition of this fact is important in interpreting HSV apoptosis results using MEF cells, especially those derived from knockout mice. The fact that primary human fibroblast and epithelial cells respond to HSV in a manner different from human cancer cells, even though they all are sensitive to environmental pro-apoptotic stimuli, emphasizes the importance of cellular pathways targeted in oncogenesis as central determinants of productive HSV replication. Future investigations in our group are focusing on defining the nature of these responses.

Together, the data presented here and in previous publications demonstrate that there are three distinct responses to HDAP. In general, most patient-derived cancer cells appear to be exquisitely sensitive to this death stimulus, primary cells derived from normal tissue are resistant, and immortalized but non-transformed cell lines may display an intermediate susceptibility. Here, we provide evidence that disruptions in the cellular apoptotic machinery probably suppress HDAP in cancer cells. Further elucidation of the exact mechanisms mediating the cell-type-dependent outcome of HSV infection will require the development of appropriate biochemical and molecular genetic systems based on our results.

We thank Elise Morton (MSSM) for expert technical cell-culture assistance and Martine Aubert, Ed Goodwin and Dan DiMaio for critical comments. These studies were supported in part by grants from the USPHS (A138873 and AI48582 to J. A. B.). M. L. N. was supported in part by USPHS Institutional Research Training Awards (AI07647 and CA088796). R. M. K. was supported in part by an Undergraduate Research Fellowship from the Howard Hughes Medical Institute to Manhattan College, Riverdale, NY, USA.