Macrophages kill human papillomavirus type 16 E6-expressing tumor cells by tumor necrosis factor alpha- and nitric oxide-dependent mechanisms

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-kappaB. We found that E1A, but not E6, blocked the TNF-alpha-induced activation of NF-kappaB, 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-kappaB 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.

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-γ (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.

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.

FIG. 3.

Role of TNF-α 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-α^−/− 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-α^−/− mice or macrophages treated with l-NAME. Results represent the means ± SEM of four separate experiments.

FIG. 4.

Relative contributions of TNF-α 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-α, 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-α and NO combination. Results represent the means ± SEM of five separate experiments.

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-γ 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.

FIG. 6.

Comparison of the expression of E1A, E6, or E7-E6 to sensitize cells to lysis by rTNF-α or NO. The figure shows cytolysis of H4, H4-E1A, H4-E6, or H4-E7-E6 cells following incubation with increasing concentrations of rTNF-α (A), DETA-NONOate (B), or rTNF-α 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-α, DETA-NONOate, or both rTNF-α and DETA-NONOate than to normal, untreated macrophages. Results represent the means ± SEM of four to five separate experiments.

FIG. 7.

Comparison of the ability of E1A, E6, or E7-E6 to block the TNF-α-induced activation of NF-κB in H4 cells. H4, E1A-, E6-, or E6-and E7-expressing cell lines were transiently transfected with a κB-luciferase (κB-luc) reporter gene construct and stimulated with 20 ng of TNF-α/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-κB is shown as relative light units and is normalized to cells not treated with TNF-α. Asterisks indicate that the TNF-α-treated cell line significantly activated (P < 0.05) NF-κB in comparison to the cell line not treated with TNF-α. Results represent the means ± SEM of three separate experiments.