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Journal of Virology, January 2005, p. 116-123, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.116-123.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Macrophages Kill Human Papillomavirus Type 16 E6-Expressing Tumor Cells by Tumor Necrosis Factor Alpha- and Nitric Oxide-Dependent Mechanisms

John M. Routes,1,2,3* Kristin Morris,2 Misoo C. Ellison,1,4 and Sharon Ryan1

Departments of Medicine,1 Integrated Department of Immunology, National Jewish Medical and Research Center and the University of Colorado Health Sciences Center,2 Cancer Center,3 Departments of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, Colorado4

Received 14 June 2004/ Accepted 20 August 2004


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ABSTRACT
 
The expression of adenovirus serotype 2 or 5 (Ad2/5) E1A sensitizes cells to killing by NK cells and activated macrophages, a property that correlates with the ability of E1A to bind the transcriptional coadaptor proteins p300-CBP. The E6 oncoproteins derived from the high-risk human papillomaviruses (HPV) interact with p300 and can complement mutant forms of E1A that cannot interact with p300 to induce cellular immortalization. Therefore, we determined if HPV type 16 (HPV16) E6 could sensitize cells to killing by macrophages and NK cells. HPV16 E6 expression sensitized human (H4 and C33A) and murine (MCA-102) cell lines to lysis by macrophages but not by NK cells. The lysis of cells that expressed E6 by macrophages was p53 independent but dependent on the production of tumor necrosis factor alpha (TNF-{alpha}) or nitric oxide (NO) by macrophages. Unlike cytolysis assays with macrophages, E6 expression did not significantly sensitize cells to lysis by the direct addition of NO or TNF-{alpha}. Like E1A, E6 has been reported to sensitize cells to lysis by TNF-{alpha} by inhibiting the TNF-{alpha}-induced activation of NF-{kappa}B. We found that E1A, but not E6, blocked the TNF-{alpha}-induced activation of NF-{kappa}B, an activity that correlated with E1A-p300 binding. In summary, Ad5 E1A and HPV16 E6 sensitized cells to lysis by macrophages. Unlike E1A, E6 did not block the ability of TNF-{alpha} to activate NF-{kappa}B or sensitize cells to lysis by NK cells, TNF-{alpha}, or NO. Thus, there appears to be a spectrum of common and unique biological activities that result as a consequence of the interaction of E6 or E1A with p300-CBP.


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INTRODUCTION
 
Adenoviruses (Ad) and human papillomaviruses (HPV) are common human pathogens that are able to transform human cells. High-risk HPV (e.g., HPV type 16 [HPV16], HPV18, and HPV31) are the etiologic agents for greater than 95% of cervical carcinomas, the second most common cancer in females worldwide (4, 53). In contrast to HPV, Ad do not appear to be oncogenic in humans (31). Ad and HPV transform cells by similar molecular mechanisms. E7 and E1A are the primary immortalizing genes of HPV and Ad, respectively. HPV E6 and Ad E1B increase the efficiency of E7- and E1A-induced immortalization and are required for complete transformation of cells. The E1A and E7 oncoproteins express homologous conserved regions, conserved region 1 (CR1) and conserved region 2 (CR2). The conserved regions of E1A and E7, which are interchangeable for immortalization function, interact with and inhibit cellular growth regulatory proteins (pRb, p107, p130, and cyclin A) (5, 20, 34, 50-52, 58, 77). Through different molecular mechanisms, the Ad E1B-55K (66, 80) and HPV E6 (55, 68) oncoproteins inhibit the function of p53. The inability of Ad to be oncogenic in humans is puzzling because the E1A and E1B oncogenes fully transform human cells (30), and cells transformed by these oncogenes form tumors in immunodeficient mice (10).

We hypothesize that one factor that influences the dissimilar oncogenicities of Ad and HPV is differences in the capacities of Ad- or HPV-transformed cells to elicit an antitumor immune response. In support of this hypothesis, we observed that tumor cells expressing Ad5 E1A are over 1,000-fold less tumorigenic than tumor cells that express HPV16 E7 or both HPV16 E7 and E6 in a syngeneic tumor model (61). The decreased tumorigenicity of tumor cells expressing E1A was dependent on a vigorous innate and adaptive immune response directed against tumor cells that express E1A. We have identified several factors that are likely to contribute to the increased immunogenicity of E1A-expressing tumor cells compared to that of E7-expressing tumor cells. E1A, but not E7, sensitizes cells to lysis by NK cells, macrophages, and the killing mechanisms utilized by these effector cells (e.g., tumor necrosis factor alpha [TNF-{alpha}], TRAIL, Fas, nitric oxide [NO], and perforin-granzyme [8, 9, 16, 39, 48, 64]). Furthermore, the sensitivity of E1A- and E7-expressing murine and human tumor cells to killing by NK cells and macrophages in vitro inversely correlates with their tumorigenicity in vivo (8, 10, 48, 60, 63, 64, 67, 78).

Based on these studies, we hypothesized that, despite many shared biological functions, molecular differences exist between the oncoproteins derived from Ad and HPV that influence the immunogenicities of Ad- compared to HPV-transformed cells. As an initial approach to this issue, we performed genetic mapping studies to determine the regions of E1A necessary to sensitize cells to lysis by NK cells and macrophages. These mapping studies demonstrated that expression of an intact CR1 region of E1A, which encompasses the p300-binding domain, was required to sensitize cells to killing by NK cells and macrophages (47). (For the purposes of this paper, we do not distinguish between p300 and the highly homologous transcriptional coactivator CBP.) The E7 oncoprotein interacts with p300. However, it lacks an N-terminal p300-binding site homologous to that of E1A (2). Studies utilizing a chimeric E1A/E7 gene that included the N terminus and CR1 (p300-binding) domain of E1A fused to CR2 and the C-terminal sequences of E7 demonstrated that the E7-p300-binding site was not equivalent to the E1A-p300-binding site in terms of sensitizing cells to lysis by either NK cells or activated macrophages (47).

The HPV16 E6 oncoprotein also interacts with p300. E1A and E6 have been reported elsewhere to interact spatially in the same functional domains of p300, and both inhibit the coactivator function of p300 (1, 21, 55). Furthermore, E6 can complement a mutant of E1A unable to bind p300 in cellular transformation assays (3). These data suggest that the biological effects of E1A and E6 on p300 function may be similar. Therefore, we determined if, like that of E1A, the expression of HPV16 E6 would sensitize cells to lysis by macrophages and NK cells. These studies indicated that E6 expression sensitized cells to lysis by macrophages but not NK cells. The macrophage-induced killing of cells expressing E6 was independent of p53 function. E1A, but not E6, sensitized cells to lysis by NO and TNF-{alpha} and to inhibited TNF-{alpha}-induced activation of NF-{kappa}B.


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MATERIALS AND METHODS
 
Cell lines. H4, a human fibrosarcoma cell line derived from HT1080, and H4-E1A (also known as P2AHT2A) were provided by S. Frisch (La Jolla Cancer Research Institute, La Jolla, Calif.). H4-E1A is an Ad5 E1A-transfected H4 cell line (27). H4-E1A-{Delta}Rb and H4-E1A-{Delta}p300 are H4 lines expressing mutant forms of E1A that fail to bind pRb and p300, respectively (48). C33A is an HPV-negative cervical cancer line. C33A and an HPV16 E6-expressing C33A line were provided by J. Huibregtse (Rutgers University, Piscataway, N.J.) (37). MCA-102 is a B6-derived, methylcholanthrene-induced sarcoma cell line and was provided by Nicholas Restifo (49) (National Institutes of Health, Bethesda, Md.). MCA-102-E1A and MCA-102-E7/E6 are MCA-102 cell lines that stably express high levels of Ad5 E1A and HPV16 E7-E6, respectively (64).

C33A, H4, and MCA-102 cell lines expressing HPV16 E6 or Ad5 E1A were derived from clones selected in G418 (Sigma-Aldrich, St. Louis, Mo.) following transfection with pLSXN16E6 or pE1A-neo, by using the Superfect transfection reagent (Qiagen, Valencia, Calif.). G418-resistant colonies were expanded and screened for the expression of Ad5 E1A or HPV16 E6 by Western (47) or Northern (62) analysis, respectively (data not shown). pLSXN16E6 was provided by Denise Galloway (Fred Hutchinson Cancer Research Center, Seattle, Wash.) (32). pE1A-neo was provided by Elizabeth Moran (Temple University, Philadelphia, Pa.) (65). All cell lines were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with antibiotics, 15 mM glucose, and 5% fetal calf serum. Cell lines were periodically tested for contamination with mycoplasma by the Mycotec assay (Bethesda Research Labs, Bethesda, Md.) and were negative.

Macrophage cytolysis assays. Macrophage cytolysis assays were performed as previously described (48). Briefly, bone marrow-derived macrophages extracted from femurs and tibias of C57BL/6 mice were grown in RPMI 1640 medium containing 10% serum and 20% granulocyte-macrophage colony-stimulating factor for approximately 7 days prior to assay. Macrophages were activated in lipopolysaccharide (LPS; 1 µg/ml; Sigma-Aldrich) and gamma interferon (IFN-{gamma}; 100 U/ml; R&D Systems, Minneapolis, Minn.) for 24 h prior to assay. Target cells were labeled with [3H]thymidine, and standard 48-h cytolysis assays were performed as described elsewhere, with the use of an optimal effector/target ratio of 50:1 (14, 48). The results shown represent the means ± standard errors of the means (SEM) of at least four separate experiments.

NK cell, TNF-{alpha}, and NO cytolysis assays. Human NK cells were isolated by negative selection with RosetteSep per the manufacturer's instructions (Stem Cell Technologies, Vancouver, British Columbia, Canada). The negatively selected cells were >90% positive for both CD16 and CD56 by fluorescence-activated cell sorting. Target cells were labeled with 51Cr and incubated with NK cells as previously described (63). DETA-NONOate [2,2'-(hydroxynitrosohydrazino)bis-ethaneamine] was used as the NO donor in the NO-dependent cytolysis assays (Calbiochem, La Jolla, Calif.) (76). Cytolysis assays for NO and TNF-{alpha} were performed as previously described (15, 48). The results shown represent the means ± SEM of at least four separate experiments. The mean percent spontaneous release from all of the target cell lines was less than 20%.

NF-{kappa}B-dependent transcription. Six-well dishes (2 x 106 cells/plate) of the parental H4 or E1A-, E6-, or E6- and E7-expressing cell lines were seeded 24 h prior to transfection. Cells were then transiently transfected with 0.5 µg of a {kappa}B-luciferase ({kappa}B-luc) reporter gene construct/well in 1 ml of serum-free DMEM/well by using Lipofectamine reagent per the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). The {kappa}B-luc construct contains three consensus NF-{kappa}B DNA binding sites from the mouse major histocompatibility complex class I (H-2 Kb) promoter (45). In addition, cells were transfected with 0.3 µg of herpes simplex virus thymidine kinase promoter-driven Renilla luciferase (pRL-TK) to control for transfection efficiency. After a 5-h incubation, cells were fed with 1 ml of DMEM-10% fetal bovine serum. Cells were incubated overnight at 37°C. Eighteen hours later cells were stimulated with 20 ng of TNF-{alpha} (R&D Systems)/ml for 6 h. Cells were subsequently washed with ice-cold phosphate-buffered saline and lysed in 200 µl of 1x Passive Lysis buffer (Promega, Madison, Wis.). Luciferase activity was assessed using the Dual Luciferase Reporter assay system (Promega). Luciferase activity was normalized to Renilla luciferase activity.

Measurement of NO. The production of NO was measured by assaying culture supernatants for the levels of nitrite, which is a stable product of NO. Bone marrow-derived macrophages were activated with LPS and IFN-{gamma} and cocultivated with H4, H4-E1A, H4-E6, and H4-E7-E6 cells for 48 h as described for the cytolysis assays (47). Nitrite in culture supernatants was measured by the Griess reaction as previously described (23).

Statistics. Data are presented as means ± SEM. The Kruskal-Wallis test was used to compare percent target cell killing among different cell lines and conditions. For pairwise comparisons, the Dunn procedure was used. Analysis of variance via a mixed effects model was also utilized with assays as random effects. Bonferroni adjustment was used to correct for multiple comparisons when appropriate. All the data analyses were carried out using SAS software (SAS Institute Inc., Cary, N.C.). A two-tailed P value of <0.05 was considered statistically significant.


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RESULTS
 
HPV16 E6 expression sensitizes tumor cells to lysis by activated macrophages but not NK cells. In order to determine whether HPV16 E6 expression could sensitize cells to lysis by NK cells and macrophages, human (C33A and H4) or murine (MCA-102) cell lines were established that stably expressed the HPV16 E6 oncogene (Materials and Methods). The MCA-102 cell lines were chosen for this study because prior studies had characterized the parental, E1A-, E7-, and E6-expressing lines in terms of their tumorigenicity in a syngeneic murine tumor model system (64). The H4 lines were selected because we had previously established H4 lines expressing mutant forms of E1A and characterized their sensitivity to lysis by NK cells and macrophages (47). C33A cells were examined to allow a comparison of the ability of E1A or E6 oncogenes to sensitize cells in a cell type that is naturally transformed by HPV.

We first determined whether the expression of Ad5 E1A, HPV16 E6, or HPV16 E7 and E6 (E7-E6) was able to sensitize H4, C33A, or MCA-102 cells to lysis by activated macrophages. As shown in Fig. 1, the expression of E1A and E6 sensitized H4, C33A, and MCA-102 cells to killing by activated macrophages. The expression of HPV16 E7 does not sensitize cells to lysis by activated macrophages (48), nor did E7 inhibit the capacity of E6 to sensitize cells to lysis by macrophages (Fig. 1).



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  		FIG. 1. Lysis of human and murine cell lines expressing Ad5 E1A, HPV16 E6, or HPV16 E7 and E6 by activated macrophages from normal mice. Standard 48-h cytolysis assays were performed using bone marrow-derived macrophages from C57BL/6 mice activated with IFN-{gamma} (100 U/ml) and LPS (1 µg/ml) (47). The figure shows lysis of human fibrosarcoma (H4) (A); HPV-negative cervical cancer cells (C33A) (B); or B6-derived, methylcholanthrene-induced sarcoma cells (MCA-102) (C) expressing Ad5 E1A, HPV16 E6, or HPV16 E7/E6 by activated macrophages. Asterisks indicate that the oncogene-expressing cell line was significantly (P < 0.05) more susceptible to lysis by macrophages than was the parental control cell line. Results represent the means ± SEM of four to seven separate experiments.

Next, we compared the ability of E6 and E1A to sensitize H4 or C33A cells to lysis by NK cells. E6 expression alone (Fig. 2A) or in combination with E7 (Fig. 2B) failed to sensitize cells to NK cell lysis. The finding that E7 and E6 expression failed to sensitize C33A cells to NK cell lysis is consistent with our previous observation on E7-E6-expressing MCA-102 cells (64). In summary, these data showed that the expression of HPV16 E6 sensitized cells to lysis by macrophages but not NK cells.



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  		FIG. 2. NK cell lysis of H4 and C33A cells that express Ad5 E1A, HPV16 E6, or HPV16 E7 and E6 by NK cells. The figure shows lysis of H4 (A) or C33A (B) tumor cells that express Ad5 E1A, HPV16 E6, or HPV16 E7-E6 by NK cells isolated from human peripheral blood mononuclear cells. Asterisks indicate that the oncogene-expressing cell line was significantly (P < 0.05) more susceptible to lysis by NK cells than was the parental control cell line at the same target cell/effector cell ratio. Results represent the means ± SEM of six separate experiments.

Macrophages kill cells expressing E1A or E6 by TNF-{alpha}- and NO-dependent mechanisms. TNF-{alpha} and NO are the principal effector mechanisms utilized by activated macrophages to kill tumor cells (9, 35, 38). The lysis of E1A-expressing cells by activated macrophages is dependent on the production of TNF-{alpha} and NO (9, 48). Therefore, we determined if macrophages kill E6-expressing cells via TNF-{alpha}, NO, or both TNF-{alpha}- and NO-dependent mechanisms. The role of TNF-{alpha} in macrophage-induced killing was examined through the use of macrophages derived from TNF-{alpha}–/– mice. Inducible NO synthase, also known as NOS2, is utilized by macrophages to generate NO. Therefore, the role of NO in macrophage killing was assessed by incubating macrophages with L-NAME (NG-monomethyl-L-arginine monoacetate), which inhibits the enzymatic activity of inducible NO synthase.

In comparison to macrophages from normal mice, macrophages unable to produce TNF-{alpha} (Fig. 3A) or NO (Fig. 3B) were impaired in their capacity to kill C33A cells expressing E1A, E6, or E7-E6. To ascertain the relative contributions of TNF and NO in the lysis of H4 cells expressing E1A-, E6-, or E7-E6, macrophage cytolysis assays were simultaneously performed using normal macrophages or macrophages lacking TNF-{alpha} or NO (Fig. 4). These data demonstrated that both NO and TNF-{alpha} contributed to the lysis of cells expressing E1A, E6, or E6 and E7 by macrophages. Macrophages lacking either TNF-{alpha} or NO exhibited an approximately 40% reduction in their capacity to kill E6-expressing cells.



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  		FIG. 3. Role of TNF-{alpha} and NO in the lysis of C33A cells expressing Ad5 E1A, HPV16 E6, or HPV16 E7/E6. The figure shows lysis of human C33A cells with macrophages isolated from TNF-{alpha}–/– mice (A) or treated with L-NAME (B), which blocks the production of NO by macrophages. Asterisks indicate that the oncogene-expressing cell line was significantly (P < 0.05) less susceptible to lysis by normal macrophages than to that by the macrophages from TNF-{alpha}–/– mice or macrophages treated with L-NAME. Results represent the means ± SEM of four separate experiments.



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  		FIG. 4. Relative contributions of TNF-{alpha} and NO to the lysis of H4 cells expressing Ad5 E1A or HPV16 E7 by macrophages. Cytolysis assays with H4, H4-E1A, H4-E6, and H4-E7/E6 as target cells were performed simultaneously using normal macrophages, macrophages lacking TNF-{alpha}, or macrophages treated with L-NAME. Asterisks indicate that the oncogene-expressing cell line was significantly (P < 0.05) less susceptible to lysis by macrophages in comparison to the TNF-{alpha} and NO combination. Results represent the means ± SEM of five separate experiments.

It is possible that the increased NO-dependent killing by macrophages of H4-E6 cells, compared to H4 cells, is due to the ability of E6 to sensitize cells to NO-induced death. Alternatively, in comparison to H4 cells, incubation of H4-E6 cells with macrophages may induce the production of higher levels of NO. In the latter case, the induction of higher levels of NO by E6-expressing cells may result in cell death without altering the intrinsic sensitivity of the cell to NO-induced killing. To address this question, NO was measured from the supernatants of cytolysis assays by using macrophages derived from normal mice or TNF-{alpha}–/– mice. The production of NO was measured by assaying culture supernatants for the levels of nitrite, which is a stable product of NO. There was no difference in the ability of H4, H4-E1A, H4-E7, or H4-E7-E6-expressing cells to induce the production of NO by macrophages (Fig. 5). Consistent with studies from our laboratory and others, macrophages derived from TNF-{alpha}–/– mice produced less NO than did macrophages derived from normal mice (44, 48; data not shown). Thus, the decreased ability of macrophages derived from TNF-{alpha}–/– mice may reflect a deficiency in NO production. In summary, these data suggested that the production of TNF-{alpha} and NO by macrophages was necessary for the optimal lysis of cells expressing E6.



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  		FIG. 5. NO release from macrophages incubated with parental (H4 and C33A), E1A-, E6-, and E7-E6-expressing cell lines. Bone marrow-derived macrophages from normal mice were activated with LPS and IFN-{gamma} and cocultivated with the indicated cell lines for 48 h. The production of NO was measured by assaying the culture supernatant for the level of nitrite, which is a stable product of NO. The results shown represent means ± SEM of three separate experiments.

E1A and E6 differ in their capacities to directly sensitize cells to lysis by rTNF-{alpha} and NO. We next tested whether the expression of E6, like E1A, would directly sensitize cells to killing by soluble NO or recombinant TNF-{alpha} (rTNF-{alpha}). H4 cells expressing E1A, E6, or E7-E6 were incubated with rTNF-{alpha}, DETA-NONOate (an NO donor), or both rTNF-{alpha} and DETA-NONOate. In agreement with prior studies (9, 18, 48), H4 cells expressing E1A were more sensitive to lysis by TNF-{alpha} (Fig. 6A) and NO (48) than parental H4 cells were (Fig. 6B). Furthermore, the combination of TNF and NO was more effective than either substance alone in killing H4-E1A cells (Fig. 6C). In contrast to E1A, E6 expression was unable to sensitize H4 cells to lysis by rTNF-{alpha} and induced a slight increase in sensitivity to lysis by NO. These data are consistent with prior studies suggesting that E6 expression fails to sensitize cells to lysis by rTNF-{alpha} (19, 26). Compared to H4-E6 cells, H4-E7-E6 cells appeared to be less sensitive to NO-induced killing. However, this difference in sensitivity was extremely small and not consistent at all concentrations of NO. Consequently, we are uncertain of the biological relevance of this finding. In summary, E1A and E6 both sensitized cells to lysis by macrophages by TNF-{alpha}- and NO-dependent mechanisms. However, unlike cells expressing E1A, cells expressing E6 were relatively resistant to lysis by the direct effects of TNF-{alpha}, NO, or both TNF-{alpha} and NO.



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  		FIG. 6. Comparison of the expression of E1A, E6, or E7-E6 to sensitize cells to lysis by rTNF-{alpha} or NO. The figure shows cytolysis of H4, H4-E1A, H4-E6, or H4-E7-E6 cells following incubation with increasing concentrations of rTNF-{alpha} (A), DETA-NONOate (B), or rTNF-{alpha} and DETA-NONOate for 24 h (C). DETA-NONOate is a compound that releases NO upon exposure to an aqueous environment. Asterisks indicate that the oncogene-expressing cell line was significantly (P < 0.05) more susceptible to lysis by rTNF-{alpha}, DETA-NONOate, or both rTNF-{alpha} and DETA-NONOate than to normal, untreated macrophages. Results represent the means ± SEM of four to five separate experiments.

E1A, but not E1A-{Delta}Rb, E1A-{Delta}p300, or E6 blocks the TNF-induced activation of the NF-{kappa}B pathway. Several studies have shown that E1A blocks TNF-{alpha}-induced, transcriptional activation of NF-{kappa}B (15, 33, 70, 71). This activity of E1A is responsible for sensitizing cells that express E1A to lysis by TNF-{alpha} (15, 56, 70). We previously showed that the capacity of E1A to sensitize H4 cells to TNF-{alpha}-dependent killing by activated macrophages correlated with the ability of E1A to bind p300-CBP, but not pRb (48). Therefore, we compared the capacities of E1A, E1A-{Delta}Rb, E1A-{Delta}p300, and E6 to block the TNF-{alpha}-induced activation of NF-{kappa}B in H4 cells. These studies demonstrated that E1A and E1A-{Delta}Rb, but not E1A-{Delta}p300, E6, or E7-E6 blocked TNF-{alpha}-induced NF-{kappa}B-dependent transcription (Fig. 7). In contrast to TNF-{alpha}, incubation of H4 cells with the NO donor DETA-NONOate failed to activate NF-{kappa}B-dependent transcription (data not shown). In summary, TNF-{alpha}-induced NF-{kappa}B-dependent transcription was blocked by E1A, but not E6. The ability of E1A to block TNF-{alpha}-induced NF-{kappa}B-dependent activation and to sensitize cells to TNF-{alpha}-induced lysis correlated with the ability of E1A to bind p300, not pRb.



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  		FIG. 7. Comparison of the ability of E1A, E6, or E7-E6 to block the TNF-{alpha}-induced activation of NF-{kappa}B in H4 cells. H4, E1A-, E6-, or E6-and E7-expressing cell lines were transiently transfected with a {kappa}B-luciferase ({kappa}B-luc) reporter gene construct and stimulated with 20 ng of TNF-{alpha}/ml for 6 h. The herpes simplex virus thymidine kinase promoter-driven Renilla luciferase (pRL-TK) was transfected to control for transfection efficiency. Luciferase activity was assessed using the Dual Luciferase Reporter assay system and normalized to Renilla luciferase activity. The activity of NF-{kappa}B is shown as relative light units and is normalized to cells not treated with TNF-{alpha}. Asterisks indicate that the TNF-{alpha}-treated cell line significantly activated (P < 0.05) NF-{kappa}B in comparison to the cell line not treated with TNF-{alpha}. Results represent the means ± SEM of three separate experiments.


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DISCUSSION
 
Results from this study demonstrated that the expression of the HPV16 E6 oncoprotein sensitized human (C33A and H4) and murine (MCA-102) tumor cells to lysis by activated macrophages but not NK cells. H4 cells, a clone of HT1080 cells, express a mutant form of p53 (17) and are resistant to lysis by macrophages (Fig. 1) (27, 48). Therefore, the ability of E6 to decrease p53 levels (79) or inhibit p53 function (81) does not contribute to the ability of E6 to sensitize cells to lysis by macrophages. Similarly, the capacity of E1A to sensitize cells to lysis by macrophages is independent of p53 function (12, 13). Macrophages killed tumor cells expressing E6 and E1A, using both TNF-{alpha}- and NO-dependent mechanisms. However, in contrast to E1A, the ability of E6 to sensitize cells to lysis by soluble rTNF-{alpha} or DETA-NONOate, a compound that releases NO upon exposure to H2O, was small and inconsistent. Perforin is the predominant killing mechanism utilized by NK cells and cytotoxic T lymphocytes (CTL) (36). Cook et al. demonstrated that expression of E1A sensitized cells to degranulation-dependent (perforin-granzyme) lysis mediated by cytolytic lymphocytes (11). Therefore, the failure of NK cells to selectively kill cells expressing E6 suggests that E6 does not sensitize cells to perforin-granzyme-dependent killing. In total these data suggest that, compared to E1A, the expression of E6 has a more restricted capacity to sensitize cells to lysis by the killing mechanisms utilized by NK cells and macrophages.

The inability of E6 to sensitize cells to lysis by soluble NO and TNF-{alpha} was unexpected in light of the finding that macrophages utilized these mechanisms to kill cells that express HPV16 E6. The production of NO in macrophages is impaired in macrophages derived from TNF-{alpha}–/– mice. Accordingly, the impaired ability of TNF-{alpha}–/– macrophages may be due to impaired generation of NO and NO-dependent killing. However, the molecular basis for the difference in the ability of E1A, compared to E6, to sensitize cells to soluble NO is unclear. These observations also suggest that macrophages utilize effector mechanisms to kill tumor cells that may not be replicated in cytolysis assays with the use of the simple addition of soluble or recombinant forms of the effector molecules.

The molecular mechanisms that enable NK cells and macrophages to selectively kill cells that express E1A are incompletely understood. Prior studies demonstrated that E1A blocks the TNF-{alpha}-induced activation of the antiapoptotic, NF-{kappa}B pathway. This activity of E1A appears to be responsible for its capacity to sensitize cells to lysis by TNF-{alpha}. There are conflicting reports on the ability of E6 to sensitize cells to lysis by rTNF (19, 25, 26, 28, 42, 43, 59, 72, 75) or to inhibit TNF-{alpha}-induced activation of the NF-{kappa}B pathway (26, 55, 72, 75). We found that the expression of E1A, but not E6, sensitized cells to rTNF-{alpha} and blocked the TNF-{alpha}-induced activation of NF-{kappa}B. Using H4 cells expressing mutants of E1A that failed to bind pRb or p300, we showed that the capacity of E1A to block the TNF-{alpha}-induced activation of NF-{kappa}B correlated with the ability of E1A to interact with p300 but not pRb. Prior observations from our laboratory with the same H4 cell lines expressing E1A-{Delta}p300 or E1A-{Delta}Rb indicated that the TNF-{alpha}-dependent killing by activated macrophages also correlated with the capacity of E1A to interact with p300. Thus, the abilities of E1A to sensitize H4 tumor cells to lysis by TNF-{alpha} and to block TNF-{alpha}-induced activation of NF-{kappa}B both correlated with E1A-p300 binding.

The molecular basis for the E1A-induced inhibition of NF-{kappa}B is also not clearly delineated. p300 and CBP are transcriptional coadaptor molecules that are essential for the optimal transcriptional activity of NF-{kappa}B, an activity inhibited by E1A. One mechanism for the ability of E1A to block the TNF-{alpha}-induced activation of the NF-{kappa}B pathway is via inhibition of the coactivator function of p300-CBP by E1A (57). E1A has also been reported to impair the degradation of I{kappa}B{alpha}, thereby blocking translocation of NF-{kappa}B to the nucleus (70). Alternatively, Cook et al. demonstrated that degradation of I{kappa}B{alpha} was not impaired by E1A and that the E1A blocked the transcriptional activity of NF-{kappa}B by a mechanism that correlated with E1A-pRb binding (15). These results illustrate the complexity of the biological effects of E1A on NF-{kappa}B activity.

The functional consequences of the interaction of p300 with viral proteins are similarly complex. p300 is a member of a family of transcriptional coadaptor molecules with several distinct functional domains (29). E1A, E7, and E6 interact with p300 in overlapping and unique regions (2, 21, 55). The spatial interaction of these viral oncoproteins with p300 partially explains their common and distinct biological effects on p300 function. For example, E7 interacts predominantly with the C/H1 domain of p300, while E1A interacts primarily with the C/H3 (TRAM) domain. The ability of E7 to interact with p300 appears to result in activities that are shared with E1A such as regulation of E2 transcriptional activity (2). In contrast, the p300-binding domains of E7 and E1A are not equivalent in their capacities to sensitize cells to lysis by NK cells and macrophages (47).

There are additional complexities apart from the spatial interactions of viral oncoproteins with p300 that influence the biological effects on p300 function. For example, although both E1A and simian virus 40 large T antigen interact with p300 in overlapping locations, large T antigen inhibits, whereas E1A enhances, the phosphorylation of p300 (22). E1A and E6 also appear to interact in the same or similar regions of p300. Our data suggested that the interaction of E1A and E6 with p300 resulted in common (induction of sensitivity to lysis by macrophages) and unique (e.g., induction of sensitivity to NK cell killing and inhibition of TNF-induced activation of NF-{kappa}B) biological effects. The ability of E1A to sensitize cells to lysis by NK cells requires expression of both the p300 binding site of E1A and a portion of exon 2 (40, 47). The expression of exon 2 of E1A can modulate cellular and viral gene expression in and of itself (54). Furthermore, amino acids encoded by exon 2 interact with proteins (e.g., CtBp) that suppress E1A-induced oncogenic transformation (6). Therefore, we hypothesize that the failure of E6 to sensitize cells to NK lysis, despite interacting with p300, is because E6 lacks a functional equivalent to exon 2 of E1A.

The ability of E1A, but not E6, to sensitize cells to NK cell lysis may have important consequences for the oncogenicity of cells that express E1A or E6. Prior studies from our laboratory with the same E1A- and E7-E6-expressing MCA-102 cell lines demonstrated that MCA-102-E1A cells were over 1,000-fold less tumorigenic than MCA-102-E7-E6 cells in syngeneic mice. These differences in tumorigenicity were due to a more effective NK cell and T-cell antitumor response directed against MCA-102 cells that express E1A (7, 41). The difference in the abilities of Ad5 E1A and HPV16 E6 to sensitize cells to lysis by NK cells but not macrophages and the potential biological consequences of this effect are reminiscent of the findings with rodent cells transformed by Ad2, Ad5, or Ad12 (7, 41). Ad12-transformed cells are oncogenic in immunocompetent rodents and are sensitive to lysis by macrophages, but not NK cells. In contrast, Ad2- or Ad5-transformed cells are oncogenic only in rodents that are depleted of NK cells or T cells and are sensitive to lysis by both NK cells and macrophages.

These studies do not exclude an important role for macrophages in the rejection of both Ad- and HPV-transformed cells. Studies of bacterial infections indicate that NK cells are necessary to activate macrophages to attain optimal bactericidal activity (69). Similarly, NK cells may interact with and prime macrophages to mediate tumor clearance. In support of this hypothesis, there is a large literature that implicates macrophages as important effectors in the rejection of cells transformed by small DNA tumor viruses (reference 41 and references therein). Furthermore, we have found that macrophages comprise a large component of the inflammatory infiltrate following the injection of MCA-102-E1A tumor cells in B6 mice (unreported observations).

There are undoubtedly other factors apart from innate immune responses that contribute to the dissimilar oncogenicities of HPV and Ad in humans. HPV-specific cytotoxic T cells in women with HPV-induced carcinomas are ineffective in mediating the clearance of E7-E6-expressing tumor cells, even when such CTL are present in significant numbers (24, 74). Studies utilizing mice transgenic for HPV16 E7 and E6 indicate that E7-specific CTL either ignore or become tolerant to keratinocytes that persistently express E7, thereby rendering these CTL ineffective in mediating antitumor immunity (46, 73). In addition, the urogenital location of HPV-induced malignancies and differences in the replicative cycle and the unique cell tropism of HPV may all contribute to the dissimilar oncogenicities of HPV and Ad.

In summary, the expression of HPV16 E6 sensitized cells to lysis by macrophages, but not NK cells. Macrophages kill E6-expressing cells by both TNF-{alpha}- and NO-dependent mechanisms. Prior studies indicate that the ability of E1A to sensitize cells to lysis by NK cells and macrophages correlates with the interaction of E1A and p300-CBP. E6 also interacts with and inhibits the function of p300-CBP. In total, these data suggest that the functional consequences of the interaction of E1A or E6 with p300-CBP are not equivalent and may result in important biological differences.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Services grant RO1-CA76491 and seed grant support funded by the University of Colorado Cancer Center (to J.M.R.) and Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Immunology (to K.M.).

We thank N. Restifo, D. Galloway, J. Huibregtse, E. Moran, and S. Frisch for reagents; S. Benedict and J. Cook for critical reading of the manuscript; and G. Cheatham for secretarial assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Phone: (303) 398-1291. Fax: (303) 398-1806. E-mail: routesj{at}njc.org. Back


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Journal of Virology, January 2005, p. 116-123, Vol. 79, No. 1
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.1.116-123.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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