|Molecular Vision 1997;
Received 15 September 1997 | Accepted 08 December 1997 | Published 17 December 1997
Inducible Apoptosis-Promoting Activity in Retinal Cell-Conditioned Medium
Gail M. Seigel, Linda Liu
Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY
Correspondence to: Gail M. Seigel, Ph.D., Box 603, Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY, 14642, Phone: (716) 273-4844, FAX: (716) 442-8766, email: GAIL_SEIGEL@URMC.ROCHESTER.EDU
Purpose: Apoptosis is implicated in the death of retinal cells during both retinal differentiation and degeneration. We sought to investigate potential diffusible retinal cell signalling factors which may be responsible for this phenomenon.
Methods: 72 hr conditioned medium was collected from death-induced R28 retinal cells undergoing serum-starvation. This conditioned medium was filtered, titrated and added to fresh cultures of R28 cells for 24 hr. Cell death was measured by trypan blue exclusion; apoptosis was evaluated by TUNEL-in situ, DNA gel electrophoresis, and observations of cell morphology.
Results: Apoptotic cell death was at least two-fold greater in retinal cultures which received death-induced cell-conditioned medium. This apoptotic activity in conditioned medium did not appear to be due to nutrient-depletion of the medium, as the control situation (72 hr conditioned medium from resistant, non-dying cells) did not produce the same effect. This diffusible apoptotic activity was heat-labile (56 °C for 45 min).
Conclusions: We have shown for the first time, in our system, that death-induced retinal cells release a diffusible, heat-labile apoptosis-promoting element. Further investigation will be necessary to determine whether this trigger of apoptosis is a toxic metabolite or an "apoptosis-promoting factor".
Apoptosis, or programmed cell death, plays a significant role in retinal degeneration, as well as retinal development. Cell loss in the developing retina has been linked with apoptotic events, and may control the distribution and number of specific cell populations in the mature neural retina (1,2). In animal models of retinal degenerative diseases, including rd/rds mice (3,4), vitiligo mice (5) and other models of retinitis pigmentosa (6), apoptosis has been identified as a pathway for photoreceptor cell death. Apoptosis of photoreceptors has also been seen in human patients after traumatic retinal detachment (7), as well as in experimental retinal detachment in cats (8). Thus, it appears that apoptosis is a common pathway for cell death occurring as part of retinal degenerative processes, as well as in retinal development. Limitations in the amount of retinal material available have slowed progress in the study of retinal apoptotic events on a cellular level. To this end, we developed immortalized retinal precursor cell lines, such as R28, which have been cultured over 100 passages, and express markers consistent with glial, as well as photoreceptor phenotypes, indicative of a pre-committment, precursor cell phenotype (9,10,11).
We can induce apoptosis in R28 cells in vitro by treatment with defined media (12; unpublished data). These metabolic challenges may mimic retinal nutrient deprivation as seen in retinal detachment (7), or the vascular compromise of diabetic retinopathy (13,14). Based on DNA fragmentation analyses and terminal deoxytransferase nick-end labelling (TUNEL)-in situ observations, the R28 retinal cell line undergoes a consistent, density-dependent apoptotic cell death in response to metabolic stress conditions that would provide an excellent model system for further study of apoptotic mechanisms relevant to retinal degeneration (12).
One exciting approach to the control of apoptotic mechanisms presumes that retinal degeneration may involve both intrinsic and extrinsic cellular mechanisms. Our in vitro system is ideal for investigations into the identity of potential environmental diffusible factors. In this report, we present evidence for the existence of a heat-labile, metabolically-inducible apoptosis-promoting activity in retinal cell-conditioned medium.
Immortalized R28 retinal precursor cells, derived from postnatal day 6 Sprague-Dawley rat, were maintained in Dulbecco Modified Eagle's Medium (DMEM) with 10% calf serum (Hyclone, Logan, UT), 1X Minimal Essential Medium (MEM) non-essential amino acids (GIBCO, Gaithersburg, MD), 1X MEM vitamins (GIBCO, Gaithersburg, MD), 0.37% sodium bicarbonate, 0.058% L-glutamine and 100 ug/ml gentamicin. For apoptosis studies, cells were treated with serumless/defined medium (DMEM/F12, 0.64% glucose, 0.148% KCl, 250 mg/ml ascorbic acid, 0.06 ng/ml progesterone, 16 mg/ml putrescine, 0.04 ng/ml sodium selenite, 0.37% sodium bicarbonate, 0.058% L-glutamine and 100 ug/ml gentamicin).
Conditioned medium was collected from cells grown for 72 hr in serumless/defined medium. The conditioned medium was passed through a 0.22 micron filter to remove dead cells and debris, then stored at -20 °C until use. Heat inactivation of conditioned medium was at 56 °C for 45 min.
Evidence of non-random DNA fragmentation was detected by agarose gel electrophoresis of DNA extracts from cells grown in serum-containing and serumless/defined medium. Non-adherent cells in the supernatant, along with trypsinized adherent cells, were utilized for each sample. For each sample of 2 x 106 cells, DNA was extracted with the Wizard Genomic DNA extraction kit (Promega, Inc., Madison, WI), and stored in the kit's DNA rehydration buffer at 4 °C until use. This extraction procedure did not allow for quantitation of DNA, but based upon cell number, approximately 1.2 µg of each DNA sample was loaded into the wells of a 1% agarose gel, with 0.5 mg/ml ethidium bromide incorporated into both the agarose gel and Tris-acetate buffer. Electrophoresis was carried out for 6 hr at 35V, 12 mA. The agarose gel was visualized and photographed using a 300 nm transilluminator (Hoefer Scientific Instruments, San Francisco, CA).
DNA fragmentation analysis in situ
Detection of DNA fragmentation in situ was visualized with the use of the Apoptag Plus Apoptosis Detection Kit (Oncor, Inc., Gaithersburg, MD) and developed with diaminobenzidine reaction product. Positive cells were counted in ten groups of 100. In some experiments, a 1:6 dilution of 0.4% Trypan blue was added to cell suspensions prior to fixation in order to assess whether cells were living or dead.
Conditioned medium from death-induced cells promotes cell death
Conditioned medium was collected from the R28 cell line after 72 hr of serumless/defined medium treatment. At 72 hr in serumless/defined medium, TUNEL-positive cells comprise greater than 60% of the population (unpublished data). By five days in serumless/defined medium, all R28 cells die. However, a 72 hr interval was chosen to ensure that there would still be metabolically active cells which could secrete factors into conditioned medium. Conditioned medium from these 72 hr death-induced cells was titrated and added to fresh R28 cultures in order to assess the death-promoting activities of death-induced cell-conditioned medium. A 24 hr timepoint was chosen to minimize background cell death known to be caused by fresh serumless/defined medium, which was used for comparison. At 24 hr, death-induced cell-conditioned medium caused cell death in a concentration-dependent manner (Figure 1). Greater than 90% of these non-adherent cells appeared dead, as evaluated by trypan blue exclusion assay.
Conditioned medium-induced cell death is apoptotic, and not due to nutrient depletion
Further analysis was necessary in order to determine whether this cell death observed was due to apoptosis. In a non-quantitative experiment, DNA gel electrophoresis of adherent and non-adherent cell DNA demonstrated the non-random DNA laddering characteristic of apoptosis, except in the serum-containing control condition (Figure 2). It was also important to address the question of nutrient depletion as a contributing factor in conditioned-medium induced cell death. Therefore, additional conditioned medium was tested: 72 hr conditioned medium from the closely-related parental culture, E1A-NR.3, from which the R28 cell line was derived. Death-resistant E1A-NR.3 cells did not exhibit significant cell death during treatment with serumless/defined medium. Conditioned medium from non-dying cells was used as a control for nutrient depletion. 72 hr conditioned media from non-dying and death-induced cells were compared with serumless/defined conditions and standard serum-containing conditions. TUNEL-in situ analysis was then used to quantitate the number of adherent apoptotic cells. As can be seen in Figure 3, conditioned medium from death-induced cells caused the greatest degree of TUNEL positivity at 24 hr. Conditioned medium from non-dying cells caused cell death at levels comparable to fresh serumless/defined medium at 24 hr. In addition, heat inactivation (56 °C for 45 min) appeared to reduce the apoptosis-promoting activity of death-induced cell conditioned medium to levels comparable with serumless/defined medium. (As an additional control, heat inactivation of fresh serumless/defined medium had no effect on the extent of apoptosis, also shown in Figure 3).
Figure 4 illustrates the morphology of the TUNEL-positive cells counted in Figure 3. Conditioned medium from death-induced cells led to a significant number of TUNEL-positive cells and apoptotic bodies in R28 cultures within 24 hr (Figure 4a). This can be seen in comparison with the control cells in Figure 4b which contained virtually no apoptotic cells. TUNEL-positive cells were present at a reduced level in other cases, including serumless/defined (Figure 4c), and heat-inactivated conditioned medium (Figure 4d). In Figure 4e, a higher magnification shows that TUNEL positive cells included full-size cells, as well as smaller apoptotic bodies.
Loss of cell adherence in conditioned medium
It is clear from the fields shown in Figure 4a that there was significant loss of adherent cells in cultures treated with death-induced cell-conditioned medium, which might mask the extent of cell death shown in Figure 3, due to detachment of dying cells which escaped TUNEL analysis. We addressed this by revisiting the number of non-adherent cells floating in the medium, which were seen to be greater than 90% dead as measured by trypan blue analysis (Figure 1). The number of dead floating cells/ml in the death-induced cell conditioned medium was at least 3-fold greater than fresh defined medium. This 3-fold difference in the number of non-adherent dead cells was even greater than the two-fold difference in the number of TUNEL positive adherent cells between the two conditions. As seen in Figure 5, when measured by trypan blue analysis, the differences between the percentage of dead cells (total of both adherent and non-adherent) under test conditions remained comparable to the TUNEL analysis shown in Figure 3. Despite significant loss of cell adherence, the majority of cells remain attached in death-induced conditioned medium at the 24 hr timepoint.
From these results, it is evident that death-induced R28 cells generate an apoptosis-promoting activity, which is heat-labile. This activity does not appear to be solely due to nutrient-depletion of the medium, as the control situation (72 hr conditioned medium from closely related non-dying cells) does not produce the same effect. Heat inactivation would also suggest against nutrient depletion, and in favor of a conditioned medium component which is neutralized by heat.
What is the nature of the cell signalling which results in apoptotic cell death? Apoptosis-promoting activity measured in our system does not depend upon cell-cell contact, but would appear to be a diffusible component released by our death-induced retinal cultures. This could be in the form of a novel apoptosis-promoting factor, or alternatively, a toxic metabolite released by death-induced cells. In our paradigm, we cannot distinguish between the possibility that the apoptosis-promoting activity is released by dying cells, or by cells not yet dying in an attempt to clear dying cells from the system as a survival strategy (hence our reference to "death-induced cell conditioned medium"). In either case, information about the triggers of retinal cell apoptosis would have many health-related implications. In human retinal degenerative disease, apoptosis is directly linked to retinal cell death as it occurs in both retinal detachment (7) and retinitis pigmentosa (15). Apoptosis is also strongly suspected as the cause of photoreceptor cell loss in other blinding, degenerative retinal diseases, including diabetic retinopathy (16,17) and macular degeneration (18). A better understanding of the triggers of retinal cell apoptosis may help identify cellular pathways and signals which would aid in the development of therapeutic strategies for all of these retinal degenerative diseases.
In a fibroblast cell culture system (19,20), intercellular induction of apoptosis was seen in transformed fibroblasts when exposed to conditioned medium from TGF-ß-treated non-transformed fibroblasts. In that paradigm, apoptotic activity was blocked by [gamma] irradiation, colchicine treatment, and thymidine treatment. In those studies, apoptosis-promoting activity was also seen to be diffusible, but dependent upon proliferation of the TGF-ß-treated non-transformed cells. In our system, apoptosis-promoting activity appears to be diffusible, yet released by metabolically challenged, death-induced retinal cells.
Naturally, much more work needs to be done in order to identify the precise nature of this inducible apoptosis-promoting activity in retinal cell line-conditioned medium. Results from these and future studies may offer insight into cell-mediated signals of apoptosis.
The authors thank Thurma McDaniel, Lois Chiu, and Kristy Coval for technical support. We appreciate the insightful comments of our anonymous reviewers. This work was supported, in part, by EY10676 (G.M.S.) and the Lucille P. Markey Charitable Trust to the University of Rochester (G.M.S.).
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