|Molecular Vision 2004;
Received 24 September 2003 | Accepted 8 April 2004 | Published 22 April 2004
Cytokine induced apoptosis in human retinoblastoma cells
Amy E. Cullinan,1
Curtis R. Brandt1,2
Departments of 1Medical Microbiology and Immunology & 2Ophthalmology and Visual Science, University of Wisconsin-Medical School, Madison, WI
Correspondence to: Curtis R. Brandt, Ph.D., University of Wisconsin,
Medical Science Center, Room 6630, 1300 University Avenue, Madison, WI,
53706; Phone: (608) 262-8054; FAX: (608) 262-0479; email:
Dr. Cullinan is now at the Department of Cell Biology, Center for Integrated Molecular Biosciences, The Scripps Research Institute, La Jolla, CA
Purpose: To determine potential anti-proliferative properties of interferon-gamma (IFN-γ) and tumor necrosis factor (TNF-α) on human retinoblastoma cells.
Methods: Fluorescent antibody staining was used to detect IFN-γ and TNF-α receptors on the cells. Y79 and Weri Rb-1 cells were exposed to IFN-γ alone, TNF-α alone, or a combination of IFN-γ and TNF-α, and apoptosis was measured by caspase 3 activation and annexin V staining. Cell cycle arrest was measured by BrdU incorporation and FACS analysis.
Results: Both cell lines expressed receptors for IFN-γ and TNF-α. There appeared to be two populations of both receptors in the Weri Rb-1 cell line. Apoptosis was induced in Y79 cells by IFN-γ, but not TNF-α, and the combination of the cytokines did not increase apoptosis above IFN-γ alone in Y79 cells. Apoptosis was induced in Weri Rb-1 cells only upon exposure to both cytokines. The cell cycle was not significantly altered in either cell line.
Conclusions: Human retinoblastoma cells respond to IFN-γ or a combination of IFN-γ and TNF-α by becoming apoptotic, but Y79 and Weri Rb-1 cells behave differently. The differential response of the two cell lines is not due to a lack of expression of IFN-γ or TNF-α receptors. The data raise the possibility that differences in apoptotic pathways exist between the two cell lines with interesting implications for the induction of apoptosis as a therapy for retinoblastoma.
Retinoblastoma is the most common intraocular malignancy in children and when left untreated is almost uniformly fatal. Traditionally, retinoblastoma has been treated by enucleation . Given that the tumor occurs in children, enucleation is particularly devastating and disfiguring. Recently, a number of therapeutic alternatives have been adopted. These include radiation, cryotherapy, chemotherapy, or various combinations of these [2-7]. These treatments are more effective with smaller tumors and are often associated with unwanted side effects, such as a higher incidence of secondary tumors, thus alternative therapeutic approaches are needed. A number of newer strategies have been recently tested, including the use of vitamin D analogs [8-11], viral therapy [12-14], and suicide gene therapy .
Another strategy actively being explored for tumor therapy is the specific induction of apoptosis. Several agents, in addition to radiation and chemotherapy have been shown to trigger apoptosis in human retinoblastoma cell lines. These include sodium butyrate , cisplatin and carboplatin , ceramide , proteosome inhibitors , topoisomerase inhibitors , arachidonic acid , retinoic acid , 8-C1-cAMP , serum deprivation, and other agents .
Tumor necrosis factor (TNF-α) and interferon-gamma (IFN-γ) are potent inducers of apoptosis and/or cell cycle inhibition and have been investigated as cancer therapies for many types of tumors [25-28]. IFN-γ is a 34 kDa homodimer that is produced by activated natural killer (NK) cells TH1-type CD4+, and CD8+ T cells. The IFN-γ receptor (IFN-γRα) is found on most nucleated cells  and consists of a high affinity ligand-binding α-chain, and the β-chain that is required for signal transduction . TNF-α was initially identified because it was observed to cause tumor necrosis [31,32]. The TNF-αreceptor-1 (TNF-R1) is found on many human cells  and most current evidence indicates that TNF-α induces apoptosis in tumor cells by ligation of receptor expressed on the cell surface, and activation of the cellular death effector machinery . TNF-α also has been reported to inhibit tumor cell proliferation by interruption of the cell cycle [35-39] as well as disrupting vascularization of solid tumors [40-42]. In humans, administration of IFN-γ and TNF-α to cancer patients has been problematic in clinical trials, mostly due to systemic toxicity of these cytokines. However, localized or intratumoral delivery of both of these inflammatory cytokines has shown promising antitumor effects with few systemic complications [25,28]. Retinoblastoma is initially restricted to the eye, thus intravitreal delivery of IFN-γ and TNF-α could result in significant antitumor effects while minimizing systemic toxicity.
In order to assess the potential of IFN-γ and TNF-α in treating retinoblastoma, it is first necessary to determine if these cytokines can inhibit the growth of retinoblastoma cells and whether any inhibitory effects are due to induction of cell cycle arrest, apoptosis, or some other mechanism. To this end, we show that IFN-γ and TNF-α can inhibit the growth of cultured Y79 and Weri Rb-1 cells, and that the primary effect is the induction of apoptosis. A second significant finding is that the two cell lines respond differently to cytokine treatment. Apoptosis was induced in Y79 cells by IFN-γ alone, while exposure to both IFN-γ and TNF-α was required to induce apoptosis in Weri Rb-1 cells. The data raise the possibility that differences in apoptotic pathways exist between the two cell lines with interesting implications for anti-apoptotic therapy for retinoblastoma.
Cell lines and cytokines
Human retinoblastoma cells (Y79 and Weri Rb-1) were obtained from ATCC and maintained in Iscove's Modified DMEM + 10% Fetal Bovine Serum (FBS) and RPMI 1640 + 10% FBS, respectively. Jurkat cells were obtained from the ATCC and were grown in RPMI 1640 + 10% FBS. Human recombinant (hr) IFN-γ and TNF-α were purchased from R & D Systems, Inc. (Minneapolis, MN). In all experiments, the final cytokine concentration was 50 ng/ml and all experiments were conducted using 10% FBS.
Cytokine receptor flow cytometry
A total of 1x106 retinoblastoma cells, grown in medium with 10% FBS, were washed with ice cold PBS + 1% FBS and incubated with 200 μg/ml of either polyclonal anti-IFN-γRα or anti-TNF-R1 antibodies (Santa Cruz Biotech, Santa Cruz, CA) or control non-specific rabbit IgG (Santa Cruz Biotech, Santa Cruz, CA) for 45 min at 4 °C in the dark. The cells were then washed with PBS + 1% FBS and were then incubated with goat anti-rabbit antibody conjugated with FITC (30 μg/ml; Jackson Immunoresearch, West Grove, PA) for 30 min at 4 °C in the dark. The cells were then washed with PBS + 1% FBS. Propidium iodide was added to a final concentration of 0.1 mg/ml, and the cells were stored on ice (30-60 min) until analyses was carried out using a FACScalibur machine (Becton-Dickson, Franklin Lakes, NJ).
Active caspase-3 staining
A total of 1x106 cells were incubated with 50 ng/ml of IFN-γ or TNF-α, or both cytokines for 72 h at 37 °C. The cells were then washed with PBS, fixed, and permeablized using the Cytofix/Cytoperm kit (Becton-Dickson, Franklin Lakes, NJ) for 20 min at RT in the dark. The cells were then washed with Permawash buffer (0.1% saponin in PBS) and stained with anti-active caspase-3 FITC (20 μg/ml, 1 h RT, Becton-Dickson, Franklin Lakes, NJ) and analyzed by flow cytometry using a FACScalibur machine. The cells were stored on ice for 30-60 min while waiting for the FACS analysis to be completed. In all apoptosis assays, positive controls consisted of Jurkat cells treated with 10 μg/ml actinomycin-D (Sigma) for 16 h at 37 °C .
Annexin V/PI analysis
A total of 1x106 treated Jurkat and human Rb cells treated with cytokines (retinoblastoma) or actinomycin D (Jurkat) as noted above, were examined for apoptosis using an annexinV-FITC detection kit (Oncogene, Inc. Boston, MA) according to manufacturer's instructions. Briefly, cells were counted, resuspended in 500 μl of cell culture medium, and annexin V-FITC plus a binding enhancer was added directly to the cells for 20 min. Propidium iodide was added as described above and the cells were analyzed with a FACScalibur machine. The cells were kept on ice (approximately 30-60 min) until the FACS analysis was completed.
BrdU staining for cell cycle analysis
A total of 3x106 retinoblastoma cells were treated with cytokines, at the concentration noted above, for 72 h in medium with 10% FBS. The cells were then pulsed with 20 μm BrdU (Sigma, St. Louis, MO) for one hour at 37 °C in 10% FBS. The cells were then fixed with cold 95% ethanol, washed with PBS and resuspended in 0.4 mg/ml pepsin in 0.1 N HCl for 30 min at room temperature (RT) to release nuclei. The nuclei were pelleted and incubated with 2 N HCl for 30 min at RT. Following neutralization with 0.1 M Na2B4O7, nuclei were pelleted and washed with PBS + 0.5% Tween-20 + 0.1% BSA. Nuclei were then stained with anti-BrdU antibody (Becton-Dickinson, Franklin Lakes, NJ) for 90 min in the dark at RT. The nuclei were then washed with PBS, pelleted and stained with FITC-labeled goat anti-mouse antibody (1:50, Becton-Dickinson) for 30 min at RT in the dark. The nuclei were washed with PBS, incubated with propidium iodide (0.1 mg/ml) and RNAse A (10 μg/ml) overnight at 4 °C, and then analyzed using a FACScalibur machine. Because of extensive aneuploidy, we chose to quantitate only the non-aneuploid portions of the FACS. The portions of the FACS analysis used for gating each population of cells were between 200 and 400 on the X-axis. The percentages, therefore, do not total 100%. However, the percentages total to over 70% for all samples thus represent the majority of cells in the population.
Cytokine receptors on retinoblastoma cells
To assess whether human retinoblastoma cells expressed receptors for IFN-γ, and TNF-α, retinoblastoma cells grown in medium containing 10% FBS were stained with antibodies specific for IFN-γRα, TNF-R1, or with non-specific rabbit IgG and then analyzed by FACS. As shown in Figure 1A, a five fold difference was seen with the IFN-γRα antibody compared to the non-specific rabbit IgG controls (geometric mean of signal intensity was 17.85 compared to 3.66 for the control) in Y79 cells. TNF-R1 was also expressed on the surface of Y79 cells (geometric mean of signal intensity was 7.85 compared to 3.66 for the control). As shown in Figure 1B, there appeared to be two populations of cells expressing both the IFN-γRα and TNF-R1 receptors in Weri Rb-1 cultures. The low expression population had four-fold and two-fold greater expression for IFN-γRα and TNF-R1 receptors, respectively, compared to the controls. The "high" expression population had 15-fold (IFN-γRα) and six-fold (TNF-R1) greater expression compared to a control non-specific antibody. These results indicate that both human retinoblastoma cell lines express receptors for both IFN-γ and TNF-α.
Cytokine induction of apoptosis
To determine if IFN-γ and TNF-α induced apoptosis in human Rb cells, caspase-3 cleavage was measured using an antibody specific for the active form. Weri Rb-1 or Y79 Retinoblastoma cells were treated with 50 ng/ml of IFN-γ, 50 ng/ml of TNF-α or 50 ng of each cytokine in culture medium containing 10% FBS for 72 h. The cells were then fixed, permeablized, stained with antibody, and analyzed by FACS. As shown in Figure 2A, active caspase was increased in Y79 cells treated with IFN-γ compared to controls (11.8% control compared to 57.1% treated). In contrast, TNF-α did not cause caspase-3 activation in Y79 cells (11.8% control compared to 12.4% treated). There appeared to be no additive or synergistic effect of the combined cytokine treatment compared to IFN-γ alone (61.2% compared to 57.1%) in the Y79 cells. The Weri Rb-1 cell line showed a different response to cytokines compared to Y79 (Figure 2B). Treatment with either IFN-γ or TNF-α alone, did not cause an increase in active caspase-3 compared to untreated cells (31.7% untreated, 37.1% IFN-γ, 34.1% TNF-α). However, combined treatment showed an increase in active caspase-3 (52.8% compared to 37.1%) suggesting that IFN-γ and TNF-α acted additively or synergistically to induce apoptosis in Weri Rb-1 cells.
To confirm the induction of apoptosis with cytokine treatment, we measured annexin V binding. The cells were treated with cytokines for 72 h in medium containing 10% FBS at a concentration of 50 ng/ml, and were stained with annexin V and propidium iodide (PI). IFN-γ treatment alone produced a similar response in Y79 cells, with 63.5% of the cells being apoptotic compared to 24.7% for the untreated cells. A typical pattern for apoptotic cells in this assay can be seen in Figure 3 with actinomycin-D treated Jurkat cells. TNF-α alone did not increase apoptosis in Y79 cells (19.7% compared to 24.7%, respectively, compared to 6.5% for untreated cells). As we found with caspase-3 activation, the combination treatment with both cytokines did not alter the number of cells undergoing apoptosis compared to IFN-γ treatment alone in Y79 cells (63.1% versus 63.5%). Cytokine treatment of Weri Rb-1 cells showed that apoptosis was induced only in response to IFN-γ and TNF-α together. A total of 68% of the cells underwent apoptosis after treatment with both cytokines, compared to 6.5% of the untreated cells (data not shown). Treatment of Weri Rb-1 cells with IFN-γ or TNF-α alone did not increase apoptosis (7.0% and 7.5%, respectively versus 6.5% for untreated cells). Taken together, the annexinV/PI results are in agreement with the caspase-3 assay and demonstrate that IFN-γ alone induces apoptosis in Y79 cells, but that a combination of IFN-γ and TNF-α is required to induce apoptosis in Weri Rb-1 cells.
Cell cycle disruption by cytokines
To determine if IFN-γ and TNF-α caused cell cycle arrest in human retinoblastoma cells, we performed BrdU incorporation assays on cytokine treated Y79 and Weri Rb-1 cells. The retinoblastoma cells were cultured for 72 h in medium containing 10% FBS and 50 ng of IFN-γ, 50 ng of TNF-α, or 50 ng of each cytokine. Figure 4A shows the BrdU profile of the Y79 cells after cytokine treatment, and Figure 4B shows the same profile for the Weri Rb-1 cells. The gated areas, representing the cell cycle phases, are shown in the first panels. Due to extensive aneuploidy of these cell lines, only the DNA content from 200-400 (on the X-axis) was quantified. Table 1 summarizes the effect of cytokines on the cell cycle in Y79 and Weri Rb-1. Neither cell line showed changes, such as a decrease in S-phase or an accumulation in G1, which would be consistent with cell cycle disruption or arrest.
Both IFN-γ and TNF-α have been investigated for use in cancer therapy. Antitumor effects have been reported, but the doses required have been found to induce significant side effects [25,28]. Tumors such as retinoblastoma, however, are amenable to treatment with local delivery of an antitumor agent. Localized delivery of IFN-γ and/or TNF-α could result in antitumor activity in the absence of systemic side effects, although localized toxicity could remain a problem. Diffusion from the tumor, into other tissues, or the circulation could occur, but the systemic effects should be greatly reduced or absent compared to parenteral administration. As an initial step in developing the possible use of IFN-γ and TNF-α for retinoblastoma therapy, we have made several important observations. First, Rb cells respond to cytokine exposure. Second, the response to cytokine exposure involves induction of apoptosis leading to death of the cells, and third, the data show that the Y79 and Weri Rb-1 cells respond differently to the two cytokines.
The finding that the two human retinoblastoma cell lines respond differently to IFN-γ and TNF-α is perhaps the most interesting conclusion of this study. Treatment of Y79 cells with IFN-γ alone effectively induced apoptosis, while Weri Rb-1 cells required exposure to both cytokines to induce an apoptotic response. Since we obtained the cells directly from ATCC and all cells used in these studies were passaged less than 10 times prior to the assays, it is unlikely that the differential responses are due to the passage history. Histologically, retinoblastoma tumors contain at least two different cell types. Some cells appear to partially differentiate and form rosettes, while others appear morphologically less differentiated. This observation raises questions as to the heterogeneity of the cells in tumors and how these populations might respond to cytokine exposure. Clearly, additional studies are needed to assess tumor cell heterogeneity and the reason for the differential response.
Both lines express the cellular receptors for IFN-γ and TNF-α (Figure 1), so the differential response is unlikely to be due to the absence of a receptor. The cytokine receptor staining data suggested that Weri Rb-1 cells might have a greater number of TNF-α receptors than Y79, at least for the "high" expressing population. This could account for a difference in response to cytokines if for example; the receptor density was insufficient in the Y79 cells. However, the fact that Y79 cells respond to IFN-γ by undergoing apoptosis indicates receptor density is sufficient for a response, at least for this cytokine. Previous studies have shown that Y79 and Weri Rb-1 cells differ with respect to photoreceptor specific gene expression. The Y79 cells express a number of non-specific proteins , whereas Weri Rb-1 cells express cone specific genes . Our finding that the two cell lines respond differently to cytokines is consistent with these results; however, it is unlikely that photoreceptor gene expression is related to differences in apoptotic responses. The observation that Weri Rb-1 cells have a population of cells expressing high levels of both receptors may also be related to differential responses of the two cell lines, but will require additional studies.
In conclusion, we have shown that human Rb cells are responsive to IFN-γ and/or TNF-α and that they respond by induction of apoptosis leading to death of the tumor cells. Interestingly, the two cell lines that were tested responded differently to cytokine exposure. The fact that IFN-γ and/or TNF-α induce tumor cell apoptosis raises the possibility that cytokine therapy may be an effective treatment for retinoblastoma.
This work was supported by a Lew R. Wasserman Award to CRB from Research to Prevent Blindness, an unrestricted grant to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, a grant to CRB from the Retina Research Foundation, Houston, TX, an award to AEC from Graduate Women in Science, and by EY07336 from the NIH. The authors would like to thank Drs. Robert Nickells and Donna Peters for helpful comments on the manuscripts.
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