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The Journal of Immunology, 2003, 170: 4119-4126.
Copyright © 2003 by The American Association of Immunologists

Adenovirus E1A, Not Human Papillomavirus E7, Sensitizes Tumor Cells to Lysis by Macrophages Through Nitric Oxide- and TNF-{alpha}-Dependent Mechanisms Despite Up-Regulation of 70-kDa Heat Shock Protein1

Tanya A. Miura*,{dagger},{ddagger}, Kristin Morris*,{dagger},{ddagger}, Sharon Ryan*, James L. Cook§ and John M. Routes2,*,{dagger},{ddagger}

Departments of * Medicine and {dagger} Immunology, National Jewish Medical and Research Center, Denver, CO 80206; {ddagger} Departments of Medicine and Immunology and the Cancer Center, University of Colorado Health Sciences Center, Denver, CO 80262; and § Departments of Medicine and Microbiology-Immunology and the Cancer Center, University of Illinois at Chicago College of Medicine, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-{alpha}, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-{alpha}, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-{alpha}. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-{alpha} and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-{alpha}, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-{alpha}, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-{alpha}, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-{alpha}-dependent mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human papillomaviruses (HPV)3 and human adenoviruses (Ad) are ubiquitous pathogens that transform mammalian cells through similar molecular mechanisms (1, 2, 3). In HPV-induced human malignancies or Ad-transformed cells, there is viral gene integration into the host genome, and expression of two viral genes (HPV, E6 and E7; Ad, E1A and E1B) is consistently found (4, 5, 6). The E1A and E7 oncoproteins contain two highly homologous, conserved regions (CR), CR1 and CR2, which serve as binding sites for cell growth regulatory proteins (1, 3, 7, 8, 9). CR function appears essential for the ability of E1A and E7 to immortalize primary cells and the CR of E1A and E7 are functionally interchangeable in transformation assays (10). Although not structurally homologous, the Ad E1B-55K and HPV E6 oncoproteins both inhibit the function of the tumor suppressor gene p53 and increase the efficiencies of E1A- and E7-induced transformation (11, 12, 13).

Despite many functional similarities between the Ad E1A and E1B and HPV E7 and E6 oncoproteins, only HPV are known to be oncogenic in humans (14, 15). The reasons for the dissimilar oncogenicities of Ad and HPV in humans are unclear. However, studies on Ad and SV40 using rodent models indicate that the competence of the innate and specific cellular immune responses to eliminate virus-transformed cells is an important factor in regulating the oncogenicity of these viruses (16, 17, 18, 19). The tumorigenicities of cells transformed by Ad and papovaviruses are inversely proportional to their capacity to be killed by NK cells and macrophages in vitro, and NK cells and macrophages are important innate effector cells involved in the rejection of cells transformed by Ad and SV40 in vivo. These observations are particularly relevant as SV40LT, the viral oncogene expressed by SV40, contains CRs (CR1, CR2) that are functionally and structurally homologous to those of E1A and E7.

Differences in the capacity of the cellular immune response to eliminate cells expressing Ad2/5 E1A or high-risk HPV16 E7 oncoproteins may also contribute to the dissimilar oncogenicities of Ad and HPV in humans. Human and murine tumor cells that express E7 or E7 and E6 oncoproteins derived from oncogenic HPV are resistant to lysis by NK cells in vitro, whereas Ad2/5 E1A-expressing tumor cells are sensitive to lysis by NK cells (20). In immunocompetent mice, syngeneic tumor cells expressing Ad5 E1A are >1000 times less tumorigenic than parental tumor cells that express HPV16 E7 or HPV16 E7 and E6 (21). These differences in tumorigenicity are entirely dependent on the increased rejection of E1A-expressing tumor cells by components of the innate and T cell-mediated antitumor immune response (21).

Previous studies showed that expression of Ad5 E1A or HPV16 E7 sensitized cells to killing by macrophages (8, 22, 23). Macrophages use several mechanisms to kill tumor cells including the production of NO, reactive oxygen intermediates (ROI), TNF-{alpha}, or Fas ligand (FasL) (24). Expression of E1A directly sensitizes cells to apoptosis by TNF-{alpha} (25, 26). Although other mechanisms may be important, TNF-{alpha} is the only effector mechanism shown to be used by macrophages to kill E1A-expressing cells (27). In contrast to E1A, expression of HPV16 E7 does not sensitize cells to killing by TNF-{alpha} (28), and the mechanisms used by macrophages to kill E7-expressing cells remain undefined.

There are several observations on NO, ROI, and FasL that may be relevant to the mechanisms by which macrophages kill E1A- or E7-expressing tumor cells. The expression of SV40LT, a viral oncoprotein functionally similar to E1A and E7, sensitizes cells to lysis by macrophages. Macrophages require the synergistic activities of both TNF-{alpha} and NO to kill cells that express SV40LT (29). ROI are formed by the membrane-associated NADPH oxidase system within macrophages and include hydrogen peroxide (H2O2), (30) hydroxyl radical (.OH), and hypochlorous acid (HOCl). Although not studied in the context as a mode of killing by macrophages, the expression of E1A sensitizes cells to lysis by H2O2 (30). Finally, the expression of E1A, but not E7, also sensitizes cells to Fas-dependent killing (28, 31). These studies suggest that macrophages may use several mechanisms to kill cells that express E1A. However, the expression of Ad5 E1A also up-regulates the expression of 70-kDa heat shock protein (Hsp70), a protein that renders tumor cells resistant to killing by TNF-{alpha}, NO, and macrophages (32, 33, 34, 35, 36, 37, 38, 39). Whether E1A can sensitize cells to lysis by macrophages, while concomitantly up-regulating Hsp70, has not been determined.

The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells expressing Ad5 E1A or HPV16 E7 oncoproteins. To accomplish this goal, we compared the ability of normal macrophages or macrophages derived from mice with genetic deficiencies in specific effector mechanisms to kill E1A- or E7-expressing murine and human tumor cells. Macrophages derived from TNF-{alpha}-/- (40) and gld mice (41) were used to examine the role of TNF-{alpha} and Fas-dependent killing. Inducible NO synthetase (iNOS) is an indispensable enzyme for the production of NO in macrophages (42). The gp91phox protein is an essential component of the NADPH oxidase system, which is necessary for the production of ROI (43). Therefore, macrophages derived from iNOS-/- and gp91phox-/- mice were used to study the roles of NO and ROI in macrophage-induced lysis of tumor cells.

We found that expression of E1A, but not E7, sensitized cells to lysis by activated macrophages. Activated macrophages killed E1A-expressing tumor cells by NO- and TNF-{alpha}-dependent mechanisms. In a manner analogous to TNF-{alpha}, E1A was found to directly sensitize tumor cells to lysis by NO. E1A up-regulated expression of Hsp70, but Hsp70 did not protect cells against lysis by TNF-{alpha}, NO, or macrophages. The biological implications of these findings are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell lines

The C57/B6-dervied, methylcholanthrene-induced sarcoma cell line MCA-102 was provided by Dr. N. Restifo (National Institutes of Health, Bethesda, MD) (44). H4, also known as HT1080, is a human fibrosarcoma cell line. MCA-102-E1A, H4-E1A (also known as P2AHT2A), and 3T3-E1A express the Ad5 E1A oncoproteins (21, 27, 45). MCA-102-E1A and H4-E1A express both the 12S and 13S forms of E1A, whereas 3T3-E1A expresses only the 13S form (27). Prior studies establish that expression of either the 13S or 12S form of E1A sensitizes cells to killing by macrophages (46). H4-E1A was provided by Dr. S. Frisch (La Jolla Cancer Institute, La Jolla, CA). H4-E7-CL1, H4-E7-CL2, MCA-102-E7-CL1, and MCA-102-E7-CL2 are HPV16 E7-transfected cell lines that express large amounts of the E7 oncoprotein (21). H4-E1A, H4-E7, MCA-102-E1A, MCA-102-E7, and 3T3-E1A all express the G418 resistance gene. 3T3 cells that express HPV16 E7 were derived from clones selected in G418 following transfection with pLSXN16E7 (provided by Dr. D. Galloway (Fred Hutchinson Cancer Research Center, Seattle, WA)) (7), which codes for G418 resistance and HPV16 E7. G418-resistant colonies were expanded and screened for the expression of HPV16 E7 by Northern analysis. 3T3-E7 cells expressed amounts of E7 mRNA comparable to those of MCA-102-E7 cells (data not shown). The same parental 3T3 cell line was used for the derivation of the 3T3-E7 and 3T3-E1A cells. Cell lines were maintained in DMEM supplemented with antibiotics, 15 mM glucose, and 5% FCS (DMEM culture medium). Cell lines were periodically tested for contamination with mycoplasma using the Mycotec assay (Bethesda Research Labs, Bethesda, MD) and were negative.

Macrophage killing assays

Normal C57/B6 mice and TNF-{alpha}, gld, and iNOS knockout mice, were obtained from The Jackson Laboratory (Bar Harbor, ME). gp91phox knockout mice were provided by Dr. A. Cooper (Colorado State University, Fort Collins, CO) (43). Bone marrow-derived macrophages were obtained from the femurs and tibias of mice and cultured in DMEM culture medium supplemented with 15% G-CSF. Confluent macrophage monolayers optimized for cytolysis assays were prepared as previously described (47). Briefly, cells were plated at a density defined in preliminary experiments to yield optimal, confluent macrophage monolayers after washing to remove nonadherent cells. This required an initial macrophage cell population plating number of 50:1 in relation to target cell number in cytotoxicity assays. All experiments were done with the same macrophage:target cell ratio and macrophage monolayer preparation technique. G-CSF was removed from the macrophage culture medium 48 h before cytolysis assays. Twenty-four hours before cytolysis assays, macrophages were further activated with IFN-{gamma} (100 U/ml; R&D Systems, Minneapolis, MN) and LPS (1 µg/ml; Sigma-Aldrich, St. Louis, MO). Flow cytometry indicated that >90% of these cells reacted with the macrophage-specific mAb F4/80 (Caltag, Burlingame, CA) (data not shown). Target cells were labeled with [3H]thymidine, and standard 48 h cytolysis assays were performed as described (22). To inhibit NO generation, cytolysis assays were performed in the presence of NG-monomethyl-L-arginine monoacetate salt (L-NAME; 1 mM), which inhibits iNOS (Calbiochem, La Jolla, CA). The results shown represent the mean ± SEM of at least four separate experiments. The mean percentage spontaneous release from all types of target cells was <20%.

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 MCA-102, MCA-102-E7, MCA-102-E1A, H4, and H4-E1A cells for 48 h as described for the cytolysis assays. Nitrite in culture supernatants was measured by the Griess reaction as previously described (48).

Western analysis

For the quantitation of Hsp70, 60-mm plates of cells were lysed in radioimmunoprecipitation analysis lysis buffer (1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 0.55% sodium deoxycholate, and 0.1% SDS). Protein concentrations were measured using Bio-Rad (Hercules, CA) protein assay. Ten micrograms of protein were separated on a 4–16% SDS-PAGE gel. Proteins were then electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% nonfat milk/PBS-Tween and incubated for 1 h with a mAb that recognizes only the inducible form of Hsp70 (Santa Cruz Biotechnology, Santa Cruz, CA). After several washes in PBS-Tween, the blot was then incubated for 1 h with HRP-conjugated anti-mouse Ab (Amersham, Piscataway, NJ). The Hsp70 protein was visualized as per the manufacturer’s instructions using the Renaissance chemiluminescence kit (DuPont-NEN, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression of E1A, not E7, sensitizes tumor cells to lysis by activated macrophages

We first compared the ability of E1A or E7 to sensitize either human (H4) or murine (MCA-102) fibrosarcoma cells to lysis by activated macrophages. Prior studies showed that H4-E1A, H4-E7, MCA-102-E1A, and MCA-102-E7 expressed high levels of the Ad5 E1A and HPV16 E7 oncoproteins (21). As shown in Fig. 1, the expression of E1A sensitized H4 or MCA-102 cells to killing by activated macrophages. In contrast, E7-expressing H4 or MCA-102 cells were no more susceptible to killing by activated macrophages than the untransfected, parental tumor cells (Fig. 1). Previous studies demonstrated the ability of HPV16 E7 to sensitize cells to killing by activated macrophages using the murine 3T3 cell line (8, 23). Therefore, to try to exclude an effect that is cell line specific, cytolysis assays were performed on Ad5 E1A- and HPV16 E7-expressing 3T3 cells. The phenotype of 3T3 cells may vary depending on the source of the cell line. Therefore, these studies used E1A- and E7-expressing 3T3 cells derived from the same 3T3 parental cell line. Consistent with our results with H4 and MCA-102 cells, expression of E1A, but not E7, sensitized 3T3 cells to killing by activated macrophages (Fig. 1B). These data indicated that, in contrast to prior reports, E7 was unable to sensitize cells to killing by activated macrophages. Therefore, we next focused on the mechanisms used by macrophages to kill E1A-expressing cells.



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  		FIGURE 1. Lysis of human and murine cell lines expressing Ad5 E1A or HPV16 E7 by activated macrophages from normal mice. Standard 48 h cytolysis assays were performed using bone marrow-derived macrophages from C57/B6 mice activated with IFN-{gamma} (100 U/ml) and LPS (1 µg/ml) (22 ). Killing of human (H4) (A) or murine (MCA-102, 3T3) (B) cell lines expressing E1A or E7 oncoproteins by activated macrophages. Results represent the mean ± SEM of four separate experiments.

 
Lysis of E1A-expressing cells by macrophages is dependent on NO and, to a lesser extent, TNF-{alpha}

Prior studies using blocking Abs to TNF-{alpha} showed that activated macrophages kill E1A-expressing tumor cells principally through TNF-{alpha}-dependent mechanisms (27). However, conclusions from such studies are limited due to the inherent technical problems with the use of blocking Abs. Therefore, we re-examined this issue by using macrophages derived from TNF-{alpha}-/- mice. As shown in Fig. 2, there was an ~15% reduction in the killing of E1A-expressing H4 or MCA-102 cells with macrophages derived from TNF-{alpha}-/- mice in comparison to that with normal macrophages. However, E1A-expressing H4 or MCA-102 cells remained significantly more sensitive than parental cells to killing by TNF-{alpha}-/- macrophages. These data suggested that, although macrophages used TNF-{alpha} to kill E1A-expressing cells, the major effector mechanism(s) were TNF-{alpha} independent. Similarly, there was a small diminution in the killing of H4 and H4-E7 cells with macrophages derived from TNF-{alpha}-/- mice in comparison to that with normal macrophages. In contrast to H4-E1A, H4-E7 cells were no more sensitive than H4 cells to killing by normal or TNF-{alpha}-/- macrophages (Fig. 2A). Thus, the expression of E7 does not appear to sensitize H4 cells to TNF-{alpha}-dependent or -independent killing by macrophages.



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  		FIGURE 2. Role of TNF-{alpha} in the lysis of human and murine cell lines expressing Ad5 E1A or HPV16 E7 by activated macrophages. Killing of human (H4) (A) or murine (MCA-102) (B) cell lines expressing E1A or E7 oncoproteins by activated macrophages derived from normal or TNF-{alpha}-/- mice. Results represent the mean ± SEM of four separate experiments.

 
Published studies indicate that the elaboration of NO is a major tumoricidal effector mechanism used by activated macrophages (49, 50). Therefore, we examined whether macrophages used NO to kill E1A-expressing cells. iNOS, also known as NOS2, is used by macrophages to generate NO. Consequently, using macrophages derived from iNOS-/- mice can assess the role of NO in the killing of tumor cells by macrophages. As shown in Fig. 3, there was an ~50% reduction in the lysis of E1A-expressing H4 and MCA-102 cells by macrophages derived from iNOS-/- mice in comparison to that by macrophages derived from normal mice.



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  		FIGURE 3. Relative contribution of TNF-{alpha} and NO to the lysis of cells expressing Ad5 E1A or HPV16 E7. Killing of human (H4) (A) or murine (MCA-102) (B) cell lines expressing E1A or E7 oncoproteins by activated macrophages derived from normal or iNOS-/- mice, or TNF-{alpha}-/- mice treated with L-NAME. Results represent the mean ± SEM of four separate experiments.

 
Next, we further quantitated the relative contributions of TNF-{alpha} and NO in the lysis of E1A-expressing tumor cells by macrophages. Cytolysis assays were performed using macrophages derived from normal mice and iNOS-/- mice (NO-independent killing), as well as macrophages derived from TNF-{alpha}-/- mice that were incubated with L-NAME, which inhibits iNOS (TNF-{alpha}- and NO-independent killing). As shown in Fig. 3, there was an ~50% reduction in killing using macrophages from iNOS-/- mice compared with normal mice. There was an additional 10–15% reduction in the killing by activated macrophages derived from TNF-{alpha}-/- mice that were incubated with L-NAME compared with macrophages derived from iNOS-/- mice. This 10–15% reduction represents contribution of TNF-{alpha} in the lysis of E1A-expressing tumor cells by activated macrophages, and is similar to the value obtained from experiments comparing tumor cell killing by macrophages from TNF-{alpha}-/- mice to that of normal mice (Fig. 2). Compared with macrophages from normal mice, macrophages unable to produce NO or both NO and TNF-{alpha} were impaired in their ability to kill H4 or H4-E7 cells. However, unlike H4-E1A, H4-E7 cells were no more sensitive than H4 cells to NO-independent or NO- and TNF-{alpha}-independent killing (Fig. 3A). Collectively, these data suggested that macrophages killed E1A-expressing tumor cells predominantly through the elaboration of NO and, to a lesser extent, TNF-{alpha}. These data also indicated that unlike E1A, expression of E7 was unable to sensitize cells to NO- and TNF-{alpha}-dependent or independent killing by macrophages.

Expression of E1A sensitizes cells to NO-dependent killing

There are several possible explanations for the NO-dependent killing of E1A-expressing cells by macrophages. E1A may directly sensitize cells to NO-induced death. Alternatively, in comparison to control or E7-expressing cells, incubation of E1A-expressing cells with macrophages may induce the production of higher levels of NO. In the latter case, the induction of higher levels of NO by E1A-expressing cells could result in cell death without altering the intrinsic sensitivity of the cell to NO-induced killing. Finally, a combination of both mechanisms may occur.

To address this question, NO was measured from the supernatants of cytolysis assays using macrophages derived from normal mice. 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 derived from normal mice were activated with LPS and IFN-{gamma} and cocultivated with MCA-102-, MCA-102-E1A-, or MCA-102-E7-expressing cells for 48 h, as described for the cytolysis assays. There was no difference in the ability of MCA-102-, MCA-102-E1A-, or MCA-102-E7-expressing cells to induce the production of NO by macrophages (Fig. 4A). In addition, the production of NO was measured using activated bone marrow-derived macrophages from normal or TNF-{alpha}-/- mice cocultivated with H4- or H4-E1A-expressing cells. H4 and H4-E1A cells did not differ in their capacity to induce the production of NO from either normal or TNF-{alpha}-deficient macrophages (Fig. 4B). However, in agreement with prior published observations, macrophages derived from TNF-{alpha}-/- mice produced less NO than macrophages derived from normal mice (Fig. 4B) (24).



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  		FIGURE 4. NO release from macrophages incubated with parental or E1A- or E7-expressing cell lines. A, Bone marrow-derived macrophages from normal mice were activated with LPS and IFN-{gamma} and cocultivated with either MCA-102, MCA-102-E1A, or MCA-102-E7 cells for 48 h. B, Production of NO by activated macrophages derived from normal and TNF-{alpha}-/- mice. Bone marrow-derived macrophages from normal or TNF-{alpha}-/- mice were activated with LPS and IFN-{gamma} and cocultivated with either H4 or H4-E1A cells for 48 h. Macrophages treated with L-NAME, which inhibits iNOS, served as a negative control for production of NO in activated macrophages. The production of NO in A and B was measured by assaying culture supernatants for the levels of nitrite, which is a stable product of NO. The results shown in A and B are representative of three separate experiments.

 
The ability of SV40LT to sensitize cells to killing by macrophages requires the synergistic activities of both TNF-{alpha} and NO (29). Prior studies established that E1A directly sensitizes cells to killing by TNF-{alpha}-/- (25, 26). We next tested whether E1A sensitized cells directly to killing by NO in the absence of macrophages. Cytolysis assays were performed on H4 and H4-E1A cells incubated with S-nitroso-N-acetylpenicillamine (SNAP), a compound that releases NO upon exposure to an aqueous environment (51). As shown in Fig. 5, H4-E1A cells were more sensitive to death when incubated with varying concentrations of SNAP than were H4 cells. In summary, these data indicated that expression of E1A directly sensitized cells to NO-dependent killing.



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  		FIGURE 5. E1A sensitizes cells to NO-dependent killing. Cytolysis of H4 and H4-E1A cells following incubation for 16 h in the presence of increasing concentrations of SNAP, an NO donor. The results shown are representative of three separate experiments.

 
Activated macrophages do not use FasL or the generation of superoxide to selectively kill E1A-expressing tumor cells

E1A sensitizes cells to lysis by Fas and H2O2 (28, 30). Therefore, it is possible that Fas or ROI act in concert with other macrophage effector mechanisms (e.g., NO and TNF-{alpha}) to efficiently kill E1A-expressing cells. Furthermore, although quantitatively small, our data showed that E1A sensitized tumor cells to macrophage killing mechanism(s) that were independent of the production of TNF-{alpha} and NO (Fig. 3). Therefore, it is possible that Fas or ROI are important effector mechanisms in the macrophage-induced killing of E1A-expressing cells. To address this possibility, we compared the killing of parental and E1A-expressing MCA-102 and H4 cells using normal macrophages, macrophages derived from gld mice (lacking FasL) or macrophages deficient in gp91phox (unable to generate ROI). In contrast to studies using macrophages derived from iNOS-/- or TNF-{alpha}-/- mice, we observed comparable killing of tumor cells with the use of macrophages derived from gld and gp91phox-/- mice compared with macrophages from normal mice (Fig. 6). Therefore, neither FasL nor ROI were necessary for the killing observed in these experiments.



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  		FIGURE 6. The role of Fas or superoxide in the lysis of human and murine cell lines expressing Ad5 E1A or HPV E7 by activated macrophages. Killing of human (H4) or murine (MCA-102) cell lines expressing E1A or E7 oncoproteins by activated macrophages derived from normal, gld (A), or gp91phox-/- (B) mice. The results shown are representative of three separate experiments.

 
Expression of E1A, but not E7, up-regulates the expression of Hsp70

The expression of Hsp70 in cells induces resistance to killing by NO, TNF-{alpha}, and activated macrophages (35, 36, 37, 38, 52, 53). Expression of E1A in cells increases production of Hsp70 (33, 39). In conjunction with our results, these studies raised the possibility that the Hsp70-induced resistance to killing by NO, TNF-{alpha}, and activated macrophages is blocked by the expression of E1A. Therefore, expression of Hsp70 protein was measured in MCA-102, MCA-102-E1A, H4, H4-E1A, and H4-E7 cells by Western analysis. In contrast to E7, the amount of Hsp70 was increased in E1A-expressing cells (Fig. 7). These data suggested that E1A mutes the antiapoptotic function of Hsp70 in tumor cells.



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  		FIGURE 7. Expression of Hsp70 in MCA-102, MCA-102-E1A, H4, H4-E1A, and H4-E7. Expression of Hsp70 was measured by Western analysis using a mAb specific for the inducible form of Hsp70.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In agreement with prior studies, we found that expression of Ad5 E1A sensitized human and murine tumor cells to killing by activated macrophages (Fig. 1). In contrast to E1A, expression of HPV16 E7 failed to sensitize nontransformed (3T3) or transformed (MCA-102, H4) human and murine cells to killing by macrophages. These data are in disagreement with studies by Matlashewski and coworkers (23) indicating that expression of HPV16 E7 sensitized 3T3 cells to killing by macrophages. Previous studies showed that macrophages injured SV40-transformed hamster cells by inhibiting mitochondrial respiration, thereby inducing a period of cytostasis. These injured SV40-transformed cells were entirely dependent on the glycolytic pathway, and hence an adequate concentration of exogenous glucose, for their survival (54, 55). Therefore, it is possible that, in previous studies that demonstrated macrophage-mediated killing of E7-expressing 3T3 cells, the concentrations of glucose were insufficient to allow glycolysis-dependent survival. Clonal differences between 3T3 cell lines may also account for this discrepancy.

Alternatively, the inability of E7 to sensitize cells to killing by macrophages may further illustrate a fundamental difference in the capacity of E1A and E7 to sensitize cells to immune effector cells and effector killing mechanisms. For example, prior studies have established that E1A sensitizes cells to killing by innate effector cells (NK cells, macrophages) and immune effector killing mechanisms (perforin, TNF-{alpha}, TRAIL and Fas) (20, 21, 22, 25, 26, 27, 30, 31, 54). In contrast, expression of E7 fails to sensitize cells to killing by NK cells, TRAIL, and Fas (20, 21, 28, 56). Although the expression of E7 has been reported to sensitize cells to killing by TNF-{alpha}, this effect is seen only with the concomitant addition of the protein synthesis inhibitor cycloheximide to the target cell (28, 56, 57). In the absence of cycloheximide, E7 fails to sensitize and may induce resistance to TNF-{alpha}-induced killing (28). In contrast, the addition of protein synthesis inhibitors is not required for the ability of E1A to induce sensitivity to any immune effector mechanisms.

Macrophages killed E1A-expressing cells predominantly through the elaboration of NO and, to a lesser extent, TNF-{alpha} (Figs. 2 and 3). This conclusion is supported by the following observations. In comparison to macrophages derived from normal mice, macrophages derived from iNOS-/- mice exhibited a ~50% reduction in their ability to kill cells that express E1A. In contrast, there was a 15% reduction in killing when macrophages were used from TNF-{alpha}-/- mice. Similarly, in comparison to macrophages that lacked NO alone, there was a 15% reduction in the lysis of cells that expressed E1A when macrophages that lacked both TNF-{alpha} and NO were used (Fig. 3).

E1A directly sensitized tumor cells to cytolysis by NO. Macrophages incubated with parental or E7- or E1A-expressing cells produced equivalent amounts of NO (Fig. 4). However, compared with parental cells, cells that expressed E1A were more sensitive to macrophage-induced killing, a process predominantly dependent on the elaboration of NO. Similarly, in comparison with parental cells, cells that expressed E1A were killed to a greater extent when incubated with the NO donor SNAP (Fig. 5). To our knowledge, this is the first demonstration that expression of E1A sensitizes cells to killing by NO.

In contrast to NO and TNF-{alpha}, neither FasL nor ROI appeared to contribute significantly to macrophage-mediated killing of E1A-expressing tumor cells. This conclusion is supported by studies using macrophages derived from gld mice or gp91phox-/- mice (Fig. 6). Thus, although expression of E1A sensitizes cells to several effector mechanisms possessed by macrophages, TNF-{alpha} and NO were the predominant mechanisms used by macrophages to kill E1A-expressing tumor cells. However, we cannot exclude the minor effects of other cytotoxic mechanisms that would be below the level of detection in these assays.

Heat shock proteins, and Hsp70 in particular, are known to be involved in the regulation of apoptosis and tumorigenesis (58, 59). Hsp70 protects tumor cells against a variety of apoptosis-inducing stimuli, including TNF-{alpha} and NO (35, 36, 37, 38, 52). Several mechanisms have been shown to be important for the antiapoptotic activity of Hsp70 and include the binding and inhibition of apoptosis-inducing factor and interaction of Hsp70 with Apaf-1, leading to inhibition of the recruitment of procaspase 9 to the apoptosome (35, 58, 59, 60). This antiapoptotic activity may contribute to the ability of Hsp70 to enhance the oncogenicity of tumor cells. For example, overexpression of Hsp70 in tumor cells decreases their sensitivity to macrophage-induced killing while increasing their tumorigenicity in syngeneic hosts (36). Likewise, inhibiting expression of Hsp70 induces tumor cell apoptosis, sensitizes cells to NO-dependent killing by macrophages, and decreases tumorigenicity (36).

In contrast to its antiapoptotic, tumorigenic effects, Hsp70 can increase the immunogenicity of endogenously expressed tumor Ags and enhance immune-mediated tumor rejection. Several mechanisms are likely to contribute to this antitumor activity of heat shock proteins including the abilities to chaperone antigenic peptides to APC, to induce the maturation of dendritic cells, to stimulate dendritic cells and macrophages to produce proinflammatory cytokines, chemokines, and NO, and to enhance tumor cell killing by NK cells (61, 62, 63, 64, 65, 66). These many activities of heat shock proteins suggest that they serve as an important link between innate and specific immunity.

E1A is a highly immunogenic oncoprotein. Infection with Ad or injection of mice with tumor cells that express E1A induces a robust innate and E1A-specific, cytotoxic T cell response (21, 67, 68, 69). It is thought that many of the immunological activities of heat shock proteins require release of endogenously expressed heat shock proteins following cell death (38, 64, 70). We hypothesize that the ability of E1A to sensitize cells to killing by innate effector cells (NK cells, macrophages), while at the same time up-regulating Hsp70, contributes to the striking immunogenicity of E1A. Although other mechanisms may also be important, we further hypothesize that these unique activities of E1A also contribute to the well-described ability of E1A to reduce the tumorigenicity of cells (21, 71, 72, 73).

Unlike E1A-expressing cells, HPV E7-expressing tumor cells are poorly immunogenic and do not readily elicit protective innate or E7-specific CTL responses in vivo (21, 74, 75, 76, 77). For example, in contrast to E1A-expressing MCA-102 tumor cells, MCA-102 tumor cells that express HPV16 E7 fail to induce a measurable NK cell or T cell response (21). Similarly, the injection of mice with E7-expressing tumor cells that are not cotransfected with the costimulatory molecule B7.1 fail to induce E7-specific CTL (77), and E7- or E6-specific CTL are inefficiently generated in women with HPV-induced cervical carcinomas (74, 75, 76). We speculate that, in contrast to E1A, the inability of E7 to sensitize cells to killing by immune effector mechanisms, along with the failure to significantly up-regulate heat shock protein expression contributes to the oncogenicity of E7-expressing, HPV-transformed cells.

In summary, expression of Ad5 E1A, but not HPV16 E7, sensitized cells to killing by activated macrophages. Expression of E1A directly sensitized cells to killing by NO. NO was also the major mechanism used by macrophages to kill tumor cells that expressed E1A. E1A also up-regulated the expression of Hsp70. However, the ability of Hsp70 to protect cells against lysis by TNF-{alpha}, NO, and macrophages was abrogated by the expression of E1A.


   Acknowledgments
 
We thank Nicholas Restifo for the MCA-102 line, Stephen Frisch for the H4 and H4-E1A cell lines, Andrea Cooper for the gp91phox-/- mice, Denise Galloway for pLSXN16E7, and Gabriele Cheatham for secretarial assistance.


   Footnotes
 
1 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.), Public Health Services Grant RO1-CA86727 and Department of the Army Grant DAMD-17-98-1-8324 (to J.L.C.), Cancer League of Colorado, National Institutes of Health Training Grant T32-AI00048, and Ferd Lawson Fellowship (to T.A.M.), and Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Immunology (to K.M.). Back

2 Address correspondence and reprint requests to Dr. John M. Routes, Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: routesj{at}njc.org Back

3 Abbreviations used in this paper: HPV, human papillomavirus; Ad, adenovirus; CR, conserved region; ROI, reactive oxygen intermediate; FasL, Fas ligand; Hsp70, 70-kDa heat shock protein; iNOS, inducible NO synthetase; L-NAME, NG-monomethyl-L-arginine monoacetate salt; SNAP, S-nitroso-N-acetylpenicillamine. Back

Received for publication October 18, 2002. Accepted for publication February 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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