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Transient transfection protects PC6-3 cells from apoptosis induced by nerve growth factor deprivation
Neuroscience 116 (2003) 23–29
LETTER TO NEUROSCIENCE TRANSIENT TRANSFECTION PROTECTS PC6-3 CELLS FROM APOPTOSIS INDUCED BY NERVE GROWTH FACTOR DEPRIVATION A. L. SCHMEHIL AND L. A. LEVIN*
death in neurotrophin-deprived neurons. To test this hypothesis, we used neuronally differentiated PC6-3 pheochromocytoma cells, a subline of the more familiar PC12 cell line, which undergo rapid and uniform apoptosis after nerve growth factor (NGF) deprivation (Pittman et al., 1993). By transiently transfecting PC6-3 cells with plasmids coding for genes that modulate ROS, we hoped to elucidate how oxidative stress could transduce a signal for neurotrophin deprivation. However, in doing these experiments we were surprised to find that the process of transfection itself protected cells from apoptosis induced by NGF deprivation, but not that induced by oxidative stress (Whittemore et al., 1994) or staurosporine (Bertrand et al., 1994). To explore this apparent neuroprotective effect, we examined the role of the transfection process in survival of these neurons after neurotrophin deprivation and other treatments that induce apoptosis.
Department of Ophthalmology and Visual Sciences and the Neuroscience Training Program, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792-4673, USA
Abstract—Some mammalian neurons undergo apoptosis after neurotrophin deprivation. We studied neuronally differentiated PC6-3 pheochromocytoma cells, which are highly dependent on nerve growth factor for survival. We found that transient transfection with green fluorescent protein or -galactosidase protected cells from apoptosis induced by nerve growth factor deprivation. Individual transfection reagent components did not produce increased viability of nerve growth factor-deprived cells. This apparent neuroprotective effect from transient transfection was specific to neurotrophin deprivation, as cells treated with H2O2 or staurosporine were not protected. To determine the mechanism of neuroprotection after transfection, the transfection status of identified groups of cells was assessed both before and after nerve growth factor deprivation. The results were consistent with a model whereby cells that are transfected but not yet expressing the transfected protein are relatively protected from nerve growth factor deprivation. We suggest that apoptosis induced by neurotrophin deprivation may interact with processes of transient transfection and expression of foreign genes in neuronal cells. Not only should these interactions be considered in transient transfection studies of neurotrophin-deprived neurons, but also their elucidation could lead to novel methods for achieving neuroprotection. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
N2 supplement was obtained from Gibco (Grand Island, NY). Other cell culture reagents were from BioWhittaker (Walkersville, MD). Fluorescein di--D-galactopyranoside (FDG) was obtained from Molecular Probes (Eugene, OR). 2-Phenylethyl--D-thiogalactopyranoside (PETG) was obtained from Acros Organics (Somerville, NJ). Murine NGF 2.5S, derived from submaxillary glands, was obtained from Alomone Laboratories, Jerusalem, Israel, and was greater than 90% pure.
Key words: PC12 cells, nerve growth factor, cell cycle.
Plasmids
EXPERIMENTAL PROCEDURES Reagents
The pEGFP-C1 green fluorescent protein (GFP) plasmid was obtained from Clontech (Palo Alto, CA). Ceruloplasmin was cloned by reverse transcription polymerase chain reaction from rat liver total RNA using published sequence (GenBank), confirmed by direct sequencing, and inserted into pEGFP-C1 to make the pCpGFP plasmid. The pSVgal plasmid was obtained from Promega (Madison, WI). Transfection-grade plasmid preparations were purified using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA).
As part of our long-term program to determine whether reactive oxygen species are a signaling mechanism in the induction of apoptosis after retinal ganglion cell axotomy (Geiger et al., 2002; Kortuem et al., 2000; Levin et al., 1996), we studied the effects of overexpression of reactive oxygen species (ROS) scavenger genes on neurotrophindeprived neurons. We hypothesized that if one or more ROS were signaling molecules in neurotrophin deprivation–induced apoptosis, then expressing proteins that modulated the levels of specific ROS would decrease cell
Cell culture PC6-3 cells (kind gift of Randall Pittman, University of Pennsylvania) were cultured in complete medium consisting of RPMI, 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml), in a humidified 5% CO2 incubator at 37°C. Cultures were grown to confluence and split every 2–5 days. To induce differentiation, cells were transferred to rat tail collagen-treated (Sigma, St. Louis, MO, USA) tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, NJ) and treated with
*Corresponding author. Tel: ⫹1-608-265-6546; fax: ⫹1-608-265-6021. E-mail address: [email protected] (L. Levine). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FDG, fluorescein di--D-galactopyranoside; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PETG, 2-phenylethyl--D-thiogalactopyranoside; ROS, reactive oxygen species.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 5 5 8 - 4
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A. L. Schmehil and L. A. Levin / Neuroscience 116 (2003) 23–29
50 ng/ml NGF in complete medium for 8 days. Half of the medium was replaced every second day. On day 8, cells were mechanically dissociated from the flask, transferred to rat tail collagentreated 24-well plates, and cultured at a concentration of 7.5⫻105 cells/ml in serum-free medium containing 1:1 (DMEM)/Ham’s F12 medium, 50 ng/ml NGF, 1% N2 serum supplement, and penicillin/ streptomycin.
Transfection At day 10 after differentiation, at which time the cells were postmitotic, cells were transfected with pEGFP-C1 or pSVgal using the Effectene Transfection Kit (Qiagen) according to the manufacturer’s directions. Briefly, 0.1 g of DNA diluted in Buffer TE was added to 4 l of Effectene reagent, 3.2 l of Enhancer reagent, and 56 l of Buffer EC. All but 350 l of medium was aspirated from the wells; 350 l of new medium was added to the transfection complexes, for a total volume of 700 l in each well. Sham-transfected cells were treated in a similar manner, with all but 350 l of medium aspirated from each well, followed by the addition of 350 l of new medium to each well. The mean (⫾S.E.M.) transfection efficiency at 72 h was 9.5⫾0.2%.
Induction of apoptosis Apoptosis was initiated at day 12 after differentiation by NGF withdrawal. Medium was aspirated from each well and replaced with 1 ml/well of 1:1 DMEM/Ham’s F12 medium, 1% N2 serum supplement, penicillin/streptomycin medium, and a 1:2000 dilution of a commercially available polyclonal rabbit antibody (immunoglobulin G fraction) against mouse 2.5S NGF (RBI/Sigma).
Assessment of transfection GFP-positive cells were identified by the presence of green fluorescence under epifluorescence using a Zeiss Axiovert 135 microscope and a fluorescein filter set. -Galactosidase-positive cells were identified as green fluorescing cells after sequential incubation in 1 mM fluorescein FDG in 1:1 phosphate-buffered saline (PBS)/H2O for 1 min at 37°C, followed by 500 M PETG in PBS at 0°C.
Assessment of apoptosis Apoptosis was assessed 21 h after NGF withdrawal using Hoechst 33258. Culture medium was replaced with 60 l of a 100 g/ml solution of Hoechst 33258 in PBS. This solution was left on cells for 20 min. Approximately 150 to 200 nuclei per well were counted under epifluorescence. Cells with condensed nuclei or nuclear condensations were scored as apoptotic.
Analysis of GFP expression during NGF deprivation A 20-gauge sterile needle was used to etch small crosshatches in the bottom of collagen-treated wells of a 24-well plate. PC6-3 cells were then plated as above. At the time of NGF deprivation, GFP⫺ and GFP⫹ cells within individual squares within the crosshatching were photographed and counted. Twenty-one hours later the individual squares were located and each cell within the square was assessed with respect to GFP status and the presence or absence of apoptosis. On average, three to six squares containing 100 to 300 cells were counted in each group. The percentage of cells transitioning from GFP⫺ to GFP⫹ was calculated by subtracting the number of cells that were GFP⫹ at the beginning of the incubation period from the number of cells that were GFP⫹ after 21 h, and dividing by the number of GFP⫹ cells at the end of 21 h.
Table 1. Increased viability of NGF-deprived PC6-3 cells after transfection with GFP Plasmid
pCpGFP pEGFP
Proportion living NGF⫹
NGF⫺/GFP⫺
NGF⫺/GFP⫹
151/151 147/148
68/169 120/234
133/149 141/151
PC6-3 cells were grown in complete medium with 50 ng/ml NGF. On day 10, cells were transfected with pCpGFP or pEGFP-C1 and the medium replaced with serum-free medium containing NGF. On day 12, some wells were deprived of NGF by replacing the NGF-containing medium with medium containing a 1:2000 dilution of antibody to NGF. Apoptosis and transfection status were assessed 21 hours later by staining with Hoechst 33258 and GFP positivity, respectively. Viability results represent the number of non-apoptotic cells divided by the total number of cells. The deprivation of NGF caused apoptosis, but there was significant survival in those cells deprived of NGF and expressing GFP, with or without co-expression of ceruloplasmin.
RESULTS Differentiated PC6-3 cells transiently transfected with green fluorescent protein are resistant to NGF deprivation PC6-3 cells were differentiated with NGF for 12 days. Cells were then either deprived of NGF or maintained in NGFcontaining medium. Cells maintained in NGF medium displayed a viability of 93.5⫾0.5%, while those deprived of NGF had a 67.8⫾3.8% viability, confirming that NGF deprivation causes differentiated PC6-3 cells to undergo apoptosis. It had previously been reported that overexpression of Cu,Zn-superoxide dismutase protects neurotrophindependent sympathetic neurons from NGF withdrawal (Greenlund et al., 1995). We hypothesized that overexpression of the ferroxidase ceruloplasmin, which inhibits the production of the ROS hydroxyl radical by oxidizing Fe⫹⫹ to Fe⫹⫹⫹, would also protect from neurotrophin deprivation (Jensen et al., 2000). To test this hypothesis, we transfected differentiated PC6-3 cells with a plasmid containing the coding sequences of Cp and GFP, the latter serving as a marker of transfection. Transfection with GFP alone was used as a control. Cells were transfected with Effectene (Qiagen) and 0.1 g/l pCpGFP or pEGFP-C1 on day 10 of NGF dependence. Forty-eight hours following transfection, apoptosis was induced in some wells by NGF deprivation. GFP⫹ and GFP⫺ cells in each well were assessed for viability with Hoechst 33258. As predicted, CpGFP⫹ cells, but not CpGFP⫺ cells, deprived of NGF had the same high viability as cells maintained in the presence of NGF (Table 1). Surprisingly, the same was true of cells transfected with the plasmid containing GFP alone, i.e. GFP⫹ cells deprived of NGF and treated with the NGF antibody maintained viability in the absence of NGF. After combination of the data from eight separate experiments, the mean survival of NGF-deprived GFP⫹ cells was 90.2⫾2.7%, significantly (P⫽0.0003) greater than that of NGF-deprived GFP⫺ cells (65.6⫾5.2%) and similar to that of GFP⫹ cells not deprived of NGF
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Fig. 1. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were incubated with various combinations of the Effectene transfection components, i.e. pEGFP-C1, Buffer EC, Effectene, and the Enhancer, and replacing the complete medium with serum-free medium. On day 12, all conditions, except a positive control containing none of the components, were deprived of NGF. Viability and transfection status were assessed 21 h later. Results are expressed as mean⫾S.E.M. percentage survival, i.e. number of nonapoptotic cells divided by the total number of cells counted. The viability of the nontransfected cells in the presence of all transfection components but in the absence of NGF was similar to that of cells deprived of NGF without the presence of the components. The viability of those cells treated with only the Effectene and Enhancer, or with just the Effectene or Enhancer, displayed a decreased viability lower than that of cells only deprived of NGF, consistent with a slight toxicity of the individual components. These results are representative of two similar experiments. NA, not applicable.
(94.6⫾1.5%). Similar results were seen when cells were transfected with identical amounts of a vector containing a different transfection marker, -galactosidase. PC6-3 cells maintained in NGF had a viability of 93.1⫾1.1%, while cells transfected with pSVgal and deprived of NGF had a viability of 92.2⫾2.9%. Those cells not transfected but deprived of NGF exhibited a lower viability of 48.4⫾1.9%. These results suggest that the GFP itself did not protect against NGF deprivation-induced apoptosis, but rather the transfection process was protective. Individual transfection components are not responsible for resistance to NGF deprivation One explanation for these results could be that one of the transfection procedure components protected against NGF deprivation-induced apoptosis. To determine whether the increased viability due to transfection with GFP was due to the effect of one of the components of the transfection complex, each component was tested separately and
in pairs, as well as all components together, as in a typical transfection. The components studied were the Effectene (cationic lipid mixture), the Enhancer (which condenses the DNA molecules), the transfection buffer, and the pEGFP-C1 plasmid. The viability of cells treated with each component separately in the absence of NGF was slightly lower than that of untreated cells, suggesting a lack of protection and perhaps slight toxicity when used separately (Fig. 1), but there was no evidence of rescue from NGF deprivation. The entire transfection procedure with the pEGFP-C1 plasmid rescued the cells, as before. This suggests that rescue of NGF-deprived cells requires the entire mechanism of transfection, and not any transfection procedure component, alone or in combination. GFP transfection does not protect against apoptosis induced by staurosporine or hydrogen peroxide The apparent protection of transiently transfected cells could be unique to NGF deprivation, or alternatively
Fig. 2. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were transfected with pEGFP-C1 and the medium was replaced with serum-free medium containing NGF. On day 12, apoptosis was induced by NGF deprivation, 100 mM staurosporine, or 430 M H2O2. Twenty-one hours later, cells were assessed for transfection status and apoptosis. Results are expressed as mean⫾S.E.M. percentage survival. GFP⫹ cells deprived of NGF showed a significant increase in viability compared with GFP⫺ cells, but not cells treated with staurosporine or H2O2. These results are representative of three similar experiments. NA, not applicable.
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could be a general protection from apoptosis induced by a variety of mechanisms. To answer this question, we studied the effect of other apoptosis inducers by treating cells with either 0.1 M staurosporine or 0.001% (0.43 mM) H2O2 to induce apoptosis. Cells treated with either staurosporine or hydrogen peroxide were maintained in NGF to prevent death from neurotrophin deprivation. Cells transfected with pEGFP-C1 and treated with staurosporine or hydrogen peroxide did not exhibit the increased viability seen with protection from NGF deprivation (Fig. 2). Therefore, although transient transfection protects PC6-3 cells from NGF deprivation, it does not protect against apoptosis induced by staurosporine or hydrogen peroxide. Mechanisms of transfection-induced increase in viability The increased number of living GFP⫹ NGF-deprived PC6-3 cells observed after transfection could result from at least three different mechanisms: (1) Neurotrophin deprivation in resistant cells could cause increased transfection efficiency or expression of the transfected protein; (2) the process of transfection could cause cells to become resistant to neurotrophin deprivation; or (3) having been transfected and expressing GFP, a cell could be more resistant to neurotrophin deprivation. To better explore which of these mechanisms might account for the increase the viability of NGF-deprived PC6-3 cells, we determined the transfection status of individual cells both immediately after NGF deprivation and 21 h following NGF deprivation. We compared the likelihood of GFP⫺ cells becoming GFP⫹ between NGF-deprived and NGF-maintained wells. We also correlated the viability of NGF-deprived cells with the likelihood that it would switch from GFP⫺ to GFP⫹. To do this, before plating several intersecting lines were etched on the bottom of each well with a sterile needle, creating individual squares in which to repeatedly observe cells in either NGF-maintained or NGF-deprived wells. At the time of NGF deprivation, cells within these squares were photographed and the number of GFP⫺ and GFP⫹ cells counted. Twenty-one hours later, the GFP status and viability of each cell within the previously identified squares were assessed. Cells which were GFP⫹ at 21 h could have been GFP⫺ or GFP⫹ at 0 h. To assess the likelihood of a cell transitioning from GFP⫺ to GFP⫹, the fraction of GFP⫹ cells which had previously been GFP⫺ was calculated. There was no difference between the NGF-deprived and NGF-maintained groups in the fraction of GFP⫹ cells which had become positive only in the previous 21 h (64⫾15% vs. 59⫾15%; P⫽0.8, n⫽6/group). There was a weak positive correlation (r⫽0.76; P⫽0.03) between the viability of a cell after NGF deprivation and the likelihood that it had become GFP⫹ during the 21 h of deprivation (Fig. 3).
DISCUSSION We used GFP as a marker of transfection in neurotrophindependent differentiated PC6-3 cells, a subline of the
Fig. 3. Boxes were etched on the bottoms of culture wells to create fields in which to observe cells. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were transfected with pEGFP-C1 and the medium was replaced with serum-free medium containing NGF. On day 12, cells were deprived of NGF and immediately photographed under epifluorescence to document the transfection status of specific fields. Twenty-one hours later, cells were incubated with Hoechst 33258 and the same fields photographed under epifluorescence to determine the transfection status and viability of cells. The number of cells transfected in each field increased during this 21-h period, while the viability of the GFP⫹ cells continued to remain significantly high, even in the absence of NGF. These results are representative of two similar experiments.
PC12 cell line (Pittman et al., 1993). Transfection of NGF-deprived cells was associated with an apparent protection of GFP⫹ cells deprived of NGF. To determine whether this phenomenon was unique to GFP, -galactosidase was used as another marker of transfection, with similar results. This implies that it is not GFP which is neuroprotective, but the transfection process. The transfection reagent components were also tested, but none individually were able to block cell death after neurotrophin deprivation. Staurosporine (Bertrand et al., 1994) and H2O2 (Whittemore et al., 1994) were used to induce apoptosis by two other methods. No apparent protection effect was seen when transfected cells were treated with either staurosporine or H2O2, suggesting that the neuroprotection is relatively specific to apoptosis induced by neurotrophin deprivation. What could explain these observations? We considered three mechanisms, and designed experiments to distinguish between them (Fig. 4): (1) removing NGF from cells resistant to the effects of neurotrophin deprivation could cause increased transfection efficiency or expression of the transfected protein; (2) the process of transfection could cause cells to become resistant to neurotrophin deprivation; or (3) transfected cells expressing the target protein could become resistant to neurotrophin deprivation. Our results showed that the fraction of GFP⫹ cells which became positive over 21 h was the same in the NGF-deprived and NGF-maintained groups, and there was a positive correlation between the viability of NGF-deprived GFP⫺ cells and the likelihood that they would become GFP⫹ during the 21 h of deprivation. These findings
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Fig. 4. Three different mechanisms proposed to explain the increased survival of transiently transfected nerve growth factor-deprived neurons. In mechanism I, it is hypothesized that nerve growth factor (NGF) deprivation promotes increased efficiency of transfection or higher levels of expression of the transfected protein. If this were the case, then higher percentages of GFP⫹ cells would be seen in the NGF-deprived condition than the NGF-maintained condition. In mechanism II, the process of being transfected prevents the cell from NGF deprivation-induced death. This would imply that higher viability would be seen in conditions where there is more transition from GFP⫺ to GFP⫹. In mechanism III, the overexpressed protein itself protects from NGF deprivation–induced death. This would entail higher viability in cells that were already GFP⫹, compared with those that transition from GFP⫺ to GFP⫹.
are consistent with a model whereby cells that are transfected and about to express GFP are relatively protected from NGF deprivation (mechanism 2). If, instead, NGF deprivation induced GFP expression (mechanism 1), then there should have been increased numbers of cells becoming GFP⫹ in the NGF-deprived group, compared with the NGF-maintained group, contrary to what was found. If the GFP protein itself were protective from NGF deprivation (mechanism 3), then cells that were GFP⫹ at the beginning of the 21-h incubation period should have been more protected from NGF deprivation than cells that became GFP⫹ during the incubation period. This would have resulted in a negative correlation between the viability of the cell after NGF deprivation and the likelihood that a cell would change from GFP⫺ to GFP⫹, contrary to the positive correlation which was found. Although this study was limited to transient transfection of PC6-3 cells in NGF deprivation-induced apoptosis, we found the same rescue effect transfecting retinal ganglion cells in primary culture (Schmehil and Levin, unpublished observation), suggesting that these findings may apply to transfection of neuronal cells in general. While GFP and -galactosidase were the only two transfection marker proteins tested, they are structurally and functionally different, and therefore it is likely that the observed results apply to other markers. Similarly, the use of two different promoters (the CMV immediate early protein and the SV40 early promoter) makes it unlikely that there was an interaction between a specific trans-acting factor affecting transgene expression and the mechanism signaling apo-
ptosis after NGF deprivation (Wiebusch and Hagemeier, 1999). Transient transfection may therefore activate a neuroprotective response within the cell, similar to the protection of axotomized retinal ganglion cells from apoptosis by transduction of an adenoviral vector alone (Kugler et al., 1999). We studied neurotrophin deprivation-induced apoptosis, as this type of cell death is vital to the understanding of apoptosis in neuronal cells. The protective effect of transfection was only seen in this type of cell death, as induction of cell death with staurosporine and H2O2 was not protected by the transfection process. This implies that the transfection process does not induce a general survival response, but rather a neuroprotective response relatively specific to neurotrophin deprivation. It is therefore likely that transient transfection affects one of the pathways for survival downstream of tyrosine receptor kinase A occupancy, e.g. phosphatidylinositol 3-kinase, Akt, or NF-B activation, or c-Jun N-terminal kinase (JNK) inhibition (Fig. 5). The fact that transient transfection was not protective of H2O2-induced apoptosis suggests that the survival pathways activated by transfection are upstream of cytochrome c release and caspase activation (Kirkland and Franklin, 2001) and do not involve JNK inhibition (Maroney et al., 1999), while the failure of transient transfection to protect against staurosporine-induced apoptosis implies that the survival response is upstream of or parallel to phosphorylation of the Akt target p70 S6 kinase (Tee and Proud, 2001). A possible interaction between transfection and neuroprotection is related to the cell cycle (Jung and Fleming-
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Fig. 5. Some of the pathways responsible for neuronal survival in the presence of nerve growth factor and death when nerve growth factor is withdrawn (䊝). Transient transfection protects from nerve growth factor deprivation, implying that it either promotes one of the survival pathways or inhibits an apoptosis-inducing pathway. Transient transfection does not protect from the apoptosis induced by staurosporine (SS) and hydrogen peroxide (H2O2), implying that their targets are downstream of the neuroprotective effect. These have been grayed out. The dashed lines indicate multiple intermediate steps. Presumably one of the pathways depicted or alternative pathways are activated by transient transfection. There is a hypothetical relationship between cell survival and the effects of transient transfection on an abortive transition from G0 to G1; see discussion.
ton, 2001; Renzing and Lane, 1995; Rodriguez and Flemington, 1999). Calcium phosphate precipitate can cause growth arrest of dividing fibroblasts in the absence of plasmid DNA (Renzing and Lane, 1995). The presumption is that calcium phosphate crystals cause cellular stress. Calcium phosphate-mediated entry of DNA into the cell induces growth arrest in G0/G1, and this can induce apoptosis in a p53-independent manner (Jung and Flemington, 2001; Rodriguez and Flemington, 1999). There were similar findings in keratinocytes transfected with a liposomalbased method (Jensen et al., 2000), and it is possible that DNA entry associated with membrane disruption and the sealing process affects the cell cycle. Even though postmitotic neurons do not progress through the cell cycle, an abortive progression beyond the G1/S transition may be a necessary element for neuronal apoptosis induced by neurotrophin deprivation, excitotoxicity, and potassium deprivation (Farinelli and Greene, 1996; Frade, 2000; Freeman et al., 1994; Kranenburg et al., 1996; Liu and Greene, 2001; Martin-Romero et al., 2000; Pabmanabhan et al., 1999), but not serum deprivation (Lindenboim et al., 1995). If transient transfection arrested the neuron in G0/G1, then this could explain the prevention of apoptosis in differentiated PC6-3 cells undergoing NGF deprivation, similar to the inhibition of apoptosis when PC12 cells are arrested at G1/S (Farinelli and Greene, 1996), cyclin-dependent ki-
nases are inhibited (Park et al., 1996), or the E2F2 transcription factor is overexpressed (Persengiev et al., 2001). Future studies should clarify whether the transfectionassociated neuroprotection of neurotrophin deprivation results from interaction with an abortive progression through the cell cycle, activation of a known survival pathway, or a novel mechanism. Acknowledgements—Supported by National Institutes of Health R01 EY12492, the Glaucoma Foundation, the Retina Research Foundation, and an unrestricted departmental grant from Research to Prevent Blindness, Inc. LAL is a Research to Prevent Blindness Dolly Green Scholar.
REFERENCES Bertrand R, Solary E, O’Connor P, Kohn KW, Pommier Y (1994) Induction of a common pathway of apoptosis by staurosporine. Exp Cell Res 211:314 –321. Farinelli SE, Greene LA (1996) Cell cycle blockers mimosine, ciclopirox, and deferoxamine prevent the death of PC12 cells and postmitotic sympathetic neurons after removal of trophic support. J Neurosci 16:1150 –1162. Frade JM (2000) Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones. J Cell Sci 113 Pt 71139 –1148. Freeman RS, Estus S, Johnson EM Jr (1994) Analysis of cell cycle-related gene expression in postmitotic neurons: selective in-
A. L. Schmehil and L. A. Levin / Neuroscience 116 (2003) 23–29 duction of Cyclin D1 during programmed cell death. Neuron 12: 343–355. Geiger LK, Kortuem KR, Alexejun C, Levin LA (2002) Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells. Neuroscience 109:635–642. Greenlund LJ, Deckwerth TL, Johnson EM (1995) Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14:303–315. Jensen UB, Petersen MS, Lund TB, Jensen TG, Bolund L (2000) Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells. Exp Dermatol 9:298 – 310. Jung EJ, Flemington EK (2001) Transfection-mediated cell synchronization: acceleration of G1-S phase transition by gamma irradiation. Biotechniques 31:1026, 1028, 1031–1024. Kirkland RA, Franklin JL (2001) Evidence for redox regulation of cytochrome C release during programmed neuronal death: antioxidant effects of protein synthesis and caspase inhibition. J Neurosci 21:1949 –1963. Kortuem K, Geiger LK, Levin LA (2000) Differential susceptibility of retinal ganglion cells to reactive oxygen species. Invest Ophthalmol Vis Sci 41:3176 –3182. Kranenburg O, van der Eb AJ, Zantema A (1996) Cyclin D1 is an essential mediator of apoptotic neuronal cell death. Embo J 15:46 – 54. Kugler S, Klocker N, Kermer P, Isenmann S, Bahr M (1999) Transduction of axotomized retinal ganglion cells by adenoviral vector administration at the optic nerve stump: an in vivo model system for the inhibition of neuronal apoptotic cell death. Gene Ther 6:1759 – 1767. Levin LA, Clark JA, Johns LK (1996) Effect of lipid peroxidation inhibition on retinal ganglion cell death. Invest Ophthalmol Vis Sci 37:2744 –2749. Lindenboim L, Diamond R, Rothenberg E, Stein R (1995) Apoptosis induced by serum deprivation of PC12 cells is not preceded by growth arrest and can occur at each phase of the cell cycle. Cancer Res 55:1242–1247. Liu DX, Greene LA (2001) Neuronal apoptosis at the G1/S cell cycle checkpoint. Cell Tissue Res 305:217–228. Maroney AC, Finn JP, Bozyczko-Coyne D, O’Kane TM, Neff NT,
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Tolkovsky AM, Park DS, Yan CY, Troy CM, Greene LA (1999) CEP-1347 (KT7515), an inhibitor of JNK activation, rescues sympathetic neurons and neuronally differentiated PC12 cells from death evoked by three distinct insults. J Neurochem 73:1901–1912. Martin-Romero FJ, Santiago-Josefat B, Correa-Bordes J, GutierrezMerino C, Fernandez-Salguero P (2000) Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition. J Mol Neurosci 15:155–165. Padmanabhan J, Park DS, Greene LA, Shelanski ML (1999) Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis. J Neurosci 19:8747–8756. Park DS, Stefanis L, Yan CY, Farinelli SE, Greene LA (1996) Ordering the cell death pathway: differential effects of BCL2, an interleukin1-converting enzyme family protease inhibitor, and other survival agents on JNK activation in serum/nerve growth factor-deprived PC12 cells. J Biol Chem 271:21898 –21905. Persengiev SP, Li J, Poulin ML, Kilpatrick DL (2001) E2F2 converts reversibly differentiated PC12 cells to an irreversible, neurotrophindependent state. Oncogene 20:5124 –5131. Pittman RN, Wang S, DiBenedetto AJ, Mills JC (1993) A system for characterizing cellular and molecular events in programmed neuronal cell death. J Neurosci 13:3669 –3680. Renzing J, Lane DP (1995) p53-dependent growth arrest following calcium phosphate-mediated transfection of murine fibroblasts. Oncogene 10:1865–1868. Rodriguez A, Flemington EK (1999) Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies. Anal Biochem 272:171–181. Tee AR, Proud CG (2001) Staurosporine inhibits phosphorylation of translational regulators linked to mTOR. Cell Death Differ 8:841– 849. Whittemore ER, Loo DT, Cotman CW (1994) Exposure to hydrogen peroxide induces cell death via apoptosis in cultured rat cortical neurons. Neuroreport 5:1485–1488. Wiebusch L, Hagemeier C (1999) Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G(1). J Virol 73: 9274 –9283. Wiebusch L, Hagemeier C (2001) The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. Embo J 20:1086 –1098.
(Accepted 29 July 2002)
LETTER TO NEUROSCIENCE TRANSIENT TRANSFECTION PROTECTS PC6-3 CELLS FROM APOPTOSIS INDUCED BY NERVE GROWTH FACTOR DEPRIVATION A. L. SCHMEHIL AND L. A. LEVIN*
death in neurotrophin-deprived neurons. To test this hypothesis, we used neuronally differentiated PC6-3 pheochromocytoma cells, a subline of the more familiar PC12 cell line, which undergo rapid and uniform apoptosis after nerve growth factor (NGF) deprivation (Pittman et al., 1993). By transiently transfecting PC6-3 cells with plasmids coding for genes that modulate ROS, we hoped to elucidate how oxidative stress could transduce a signal for neurotrophin deprivation. However, in doing these experiments we were surprised to find that the process of transfection itself protected cells from apoptosis induced by NGF deprivation, but not that induced by oxidative stress (Whittemore et al., 1994) or staurosporine (Bertrand et al., 1994). To explore this apparent neuroprotective effect, we examined the role of the transfection process in survival of these neurons after neurotrophin deprivation and other treatments that induce apoptosis.
Department of Ophthalmology and Visual Sciences and the Neuroscience Training Program, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792-4673, USA
Abstract—Some mammalian neurons undergo apoptosis after neurotrophin deprivation. We studied neuronally differentiated PC6-3 pheochromocytoma cells, which are highly dependent on nerve growth factor for survival. We found that transient transfection with green fluorescent protein or -galactosidase protected cells from apoptosis induced by nerve growth factor deprivation. Individual transfection reagent components did not produce increased viability of nerve growth factor-deprived cells. This apparent neuroprotective effect from transient transfection was specific to neurotrophin deprivation, as cells treated with H2O2 or staurosporine were not protected. To determine the mechanism of neuroprotection after transfection, the transfection status of identified groups of cells was assessed both before and after nerve growth factor deprivation. The results were consistent with a model whereby cells that are transfected but not yet expressing the transfected protein are relatively protected from nerve growth factor deprivation. We suggest that apoptosis induced by neurotrophin deprivation may interact with processes of transient transfection and expression of foreign genes in neuronal cells. Not only should these interactions be considered in transient transfection studies of neurotrophin-deprived neurons, but also their elucidation could lead to novel methods for achieving neuroprotection. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
N2 supplement was obtained from Gibco (Grand Island, NY). Other cell culture reagents were from BioWhittaker (Walkersville, MD). Fluorescein di--D-galactopyranoside (FDG) was obtained from Molecular Probes (Eugene, OR). 2-Phenylethyl--D-thiogalactopyranoside (PETG) was obtained from Acros Organics (Somerville, NJ). Murine NGF 2.5S, derived from submaxillary glands, was obtained from Alomone Laboratories, Jerusalem, Israel, and was greater than 90% pure.
Key words: PC12 cells, nerve growth factor, cell cycle.
Plasmids
EXPERIMENTAL PROCEDURES Reagents
The pEGFP-C1 green fluorescent protein (GFP) plasmid was obtained from Clontech (Palo Alto, CA). Ceruloplasmin was cloned by reverse transcription polymerase chain reaction from rat liver total RNA using published sequence (GenBank), confirmed by direct sequencing, and inserted into pEGFP-C1 to make the pCpGFP plasmid. The pSVgal plasmid was obtained from Promega (Madison, WI). Transfection-grade plasmid preparations were purified using an EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA).
As part of our long-term program to determine whether reactive oxygen species are a signaling mechanism in the induction of apoptosis after retinal ganglion cell axotomy (Geiger et al., 2002; Kortuem et al., 2000; Levin et al., 1996), we studied the effects of overexpression of reactive oxygen species (ROS) scavenger genes on neurotrophindeprived neurons. We hypothesized that if one or more ROS were signaling molecules in neurotrophin deprivation–induced apoptosis, then expressing proteins that modulated the levels of specific ROS would decrease cell
Cell culture PC6-3 cells (kind gift of Randall Pittman, University of Pennsylvania) were cultured in complete medium consisting of RPMI, 10% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml), in a humidified 5% CO2 incubator at 37°C. Cultures were grown to confluence and split every 2–5 days. To induce differentiation, cells were transferred to rat tail collagen-treated (Sigma, St. Louis, MO, USA) tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, NJ) and treated with
*Corresponding author. Tel: ⫹1-608-265-6546; fax: ⫹1-608-265-6021. E-mail address: [email protected] (L. Levine). Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FDG, fluorescein di--D-galactopyranoside; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PETG, 2-phenylethyl--D-thiogalactopyranoside; ROS, reactive oxygen species.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 5 5 8 - 4
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50 ng/ml NGF in complete medium for 8 days. Half of the medium was replaced every second day. On day 8, cells were mechanically dissociated from the flask, transferred to rat tail collagentreated 24-well plates, and cultured at a concentration of 7.5⫻105 cells/ml in serum-free medium containing 1:1 (DMEM)/Ham’s F12 medium, 50 ng/ml NGF, 1% N2 serum supplement, and penicillin/ streptomycin.
Transfection At day 10 after differentiation, at which time the cells were postmitotic, cells were transfected with pEGFP-C1 or pSVgal using the Effectene Transfection Kit (Qiagen) according to the manufacturer’s directions. Briefly, 0.1 g of DNA diluted in Buffer TE was added to 4 l of Effectene reagent, 3.2 l of Enhancer reagent, and 56 l of Buffer EC. All but 350 l of medium was aspirated from the wells; 350 l of new medium was added to the transfection complexes, for a total volume of 700 l in each well. Sham-transfected cells were treated in a similar manner, with all but 350 l of medium aspirated from each well, followed by the addition of 350 l of new medium to each well. The mean (⫾S.E.M.) transfection efficiency at 72 h was 9.5⫾0.2%.
Induction of apoptosis Apoptosis was initiated at day 12 after differentiation by NGF withdrawal. Medium was aspirated from each well and replaced with 1 ml/well of 1:1 DMEM/Ham’s F12 medium, 1% N2 serum supplement, penicillin/streptomycin medium, and a 1:2000 dilution of a commercially available polyclonal rabbit antibody (immunoglobulin G fraction) against mouse 2.5S NGF (RBI/Sigma).
Assessment of transfection GFP-positive cells were identified by the presence of green fluorescence under epifluorescence using a Zeiss Axiovert 135 microscope and a fluorescein filter set. -Galactosidase-positive cells were identified as green fluorescing cells after sequential incubation in 1 mM fluorescein FDG in 1:1 phosphate-buffered saline (PBS)/H2O for 1 min at 37°C, followed by 500 M PETG in PBS at 0°C.
Assessment of apoptosis Apoptosis was assessed 21 h after NGF withdrawal using Hoechst 33258. Culture medium was replaced with 60 l of a 100 g/ml solution of Hoechst 33258 in PBS. This solution was left on cells for 20 min. Approximately 150 to 200 nuclei per well were counted under epifluorescence. Cells with condensed nuclei or nuclear condensations were scored as apoptotic.
Analysis of GFP expression during NGF deprivation A 20-gauge sterile needle was used to etch small crosshatches in the bottom of collagen-treated wells of a 24-well plate. PC6-3 cells were then plated as above. At the time of NGF deprivation, GFP⫺ and GFP⫹ cells within individual squares within the crosshatching were photographed and counted. Twenty-one hours later the individual squares were located and each cell within the square was assessed with respect to GFP status and the presence or absence of apoptosis. On average, three to six squares containing 100 to 300 cells were counted in each group. The percentage of cells transitioning from GFP⫺ to GFP⫹ was calculated by subtracting the number of cells that were GFP⫹ at the beginning of the incubation period from the number of cells that were GFP⫹ after 21 h, and dividing by the number of GFP⫹ cells at the end of 21 h.
Table 1. Increased viability of NGF-deprived PC6-3 cells after transfection with GFP Plasmid
pCpGFP pEGFP
Proportion living NGF⫹
NGF⫺/GFP⫺
NGF⫺/GFP⫹
151/151 147/148
68/169 120/234
133/149 141/151
PC6-3 cells were grown in complete medium with 50 ng/ml NGF. On day 10, cells were transfected with pCpGFP or pEGFP-C1 and the medium replaced with serum-free medium containing NGF. On day 12, some wells were deprived of NGF by replacing the NGF-containing medium with medium containing a 1:2000 dilution of antibody to NGF. Apoptosis and transfection status were assessed 21 hours later by staining with Hoechst 33258 and GFP positivity, respectively. Viability results represent the number of non-apoptotic cells divided by the total number of cells. The deprivation of NGF caused apoptosis, but there was significant survival in those cells deprived of NGF and expressing GFP, with or without co-expression of ceruloplasmin.
RESULTS Differentiated PC6-3 cells transiently transfected with green fluorescent protein are resistant to NGF deprivation PC6-3 cells were differentiated with NGF for 12 days. Cells were then either deprived of NGF or maintained in NGFcontaining medium. Cells maintained in NGF medium displayed a viability of 93.5⫾0.5%, while those deprived of NGF had a 67.8⫾3.8% viability, confirming that NGF deprivation causes differentiated PC6-3 cells to undergo apoptosis. It had previously been reported that overexpression of Cu,Zn-superoxide dismutase protects neurotrophindependent sympathetic neurons from NGF withdrawal (Greenlund et al., 1995). We hypothesized that overexpression of the ferroxidase ceruloplasmin, which inhibits the production of the ROS hydroxyl radical by oxidizing Fe⫹⫹ to Fe⫹⫹⫹, would also protect from neurotrophin deprivation (Jensen et al., 2000). To test this hypothesis, we transfected differentiated PC6-3 cells with a plasmid containing the coding sequences of Cp and GFP, the latter serving as a marker of transfection. Transfection with GFP alone was used as a control. Cells were transfected with Effectene (Qiagen) and 0.1 g/l pCpGFP or pEGFP-C1 on day 10 of NGF dependence. Forty-eight hours following transfection, apoptosis was induced in some wells by NGF deprivation. GFP⫹ and GFP⫺ cells in each well were assessed for viability with Hoechst 33258. As predicted, CpGFP⫹ cells, but not CpGFP⫺ cells, deprived of NGF had the same high viability as cells maintained in the presence of NGF (Table 1). Surprisingly, the same was true of cells transfected with the plasmid containing GFP alone, i.e. GFP⫹ cells deprived of NGF and treated with the NGF antibody maintained viability in the absence of NGF. After combination of the data from eight separate experiments, the mean survival of NGF-deprived GFP⫹ cells was 90.2⫾2.7%, significantly (P⫽0.0003) greater than that of NGF-deprived GFP⫺ cells (65.6⫾5.2%) and similar to that of GFP⫹ cells not deprived of NGF
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Fig. 1. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were incubated with various combinations of the Effectene transfection components, i.e. pEGFP-C1, Buffer EC, Effectene, and the Enhancer, and replacing the complete medium with serum-free medium. On day 12, all conditions, except a positive control containing none of the components, were deprived of NGF. Viability and transfection status were assessed 21 h later. Results are expressed as mean⫾S.E.M. percentage survival, i.e. number of nonapoptotic cells divided by the total number of cells counted. The viability of the nontransfected cells in the presence of all transfection components but in the absence of NGF was similar to that of cells deprived of NGF without the presence of the components. The viability of those cells treated with only the Effectene and Enhancer, or with just the Effectene or Enhancer, displayed a decreased viability lower than that of cells only deprived of NGF, consistent with a slight toxicity of the individual components. These results are representative of two similar experiments. NA, not applicable.
(94.6⫾1.5%). Similar results were seen when cells were transfected with identical amounts of a vector containing a different transfection marker, -galactosidase. PC6-3 cells maintained in NGF had a viability of 93.1⫾1.1%, while cells transfected with pSVgal and deprived of NGF had a viability of 92.2⫾2.9%. Those cells not transfected but deprived of NGF exhibited a lower viability of 48.4⫾1.9%. These results suggest that the GFP itself did not protect against NGF deprivation-induced apoptosis, but rather the transfection process was protective. Individual transfection components are not responsible for resistance to NGF deprivation One explanation for these results could be that one of the transfection procedure components protected against NGF deprivation-induced apoptosis. To determine whether the increased viability due to transfection with GFP was due to the effect of one of the components of the transfection complex, each component was tested separately and
in pairs, as well as all components together, as in a typical transfection. The components studied were the Effectene (cationic lipid mixture), the Enhancer (which condenses the DNA molecules), the transfection buffer, and the pEGFP-C1 plasmid. The viability of cells treated with each component separately in the absence of NGF was slightly lower than that of untreated cells, suggesting a lack of protection and perhaps slight toxicity when used separately (Fig. 1), but there was no evidence of rescue from NGF deprivation. The entire transfection procedure with the pEGFP-C1 plasmid rescued the cells, as before. This suggests that rescue of NGF-deprived cells requires the entire mechanism of transfection, and not any transfection procedure component, alone or in combination. GFP transfection does not protect against apoptosis induced by staurosporine or hydrogen peroxide The apparent protection of transiently transfected cells could be unique to NGF deprivation, or alternatively
Fig. 2. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were transfected with pEGFP-C1 and the medium was replaced with serum-free medium containing NGF. On day 12, apoptosis was induced by NGF deprivation, 100 mM staurosporine, or 430 M H2O2. Twenty-one hours later, cells were assessed for transfection status and apoptosis. Results are expressed as mean⫾S.E.M. percentage survival. GFP⫹ cells deprived of NGF showed a significant increase in viability compared with GFP⫺ cells, but not cells treated with staurosporine or H2O2. These results are representative of three similar experiments. NA, not applicable.
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could be a general protection from apoptosis induced by a variety of mechanisms. To answer this question, we studied the effect of other apoptosis inducers by treating cells with either 0.1 M staurosporine or 0.001% (0.43 mM) H2O2 to induce apoptosis. Cells treated with either staurosporine or hydrogen peroxide were maintained in NGF to prevent death from neurotrophin deprivation. Cells transfected with pEGFP-C1 and treated with staurosporine or hydrogen peroxide did not exhibit the increased viability seen with protection from NGF deprivation (Fig. 2). Therefore, although transient transfection protects PC6-3 cells from NGF deprivation, it does not protect against apoptosis induced by staurosporine or hydrogen peroxide. Mechanisms of transfection-induced increase in viability The increased number of living GFP⫹ NGF-deprived PC6-3 cells observed after transfection could result from at least three different mechanisms: (1) Neurotrophin deprivation in resistant cells could cause increased transfection efficiency or expression of the transfected protein; (2) the process of transfection could cause cells to become resistant to neurotrophin deprivation; or (3) having been transfected and expressing GFP, a cell could be more resistant to neurotrophin deprivation. To better explore which of these mechanisms might account for the increase the viability of NGF-deprived PC6-3 cells, we determined the transfection status of individual cells both immediately after NGF deprivation and 21 h following NGF deprivation. We compared the likelihood of GFP⫺ cells becoming GFP⫹ between NGF-deprived and NGF-maintained wells. We also correlated the viability of NGF-deprived cells with the likelihood that it would switch from GFP⫺ to GFP⫹. To do this, before plating several intersecting lines were etched on the bottom of each well with a sterile needle, creating individual squares in which to repeatedly observe cells in either NGF-maintained or NGF-deprived wells. At the time of NGF deprivation, cells within these squares were photographed and the number of GFP⫺ and GFP⫹ cells counted. Twenty-one hours later, the GFP status and viability of each cell within the previously identified squares were assessed. Cells which were GFP⫹ at 21 h could have been GFP⫺ or GFP⫹ at 0 h. To assess the likelihood of a cell transitioning from GFP⫺ to GFP⫹, the fraction of GFP⫹ cells which had previously been GFP⫺ was calculated. There was no difference between the NGF-deprived and NGF-maintained groups in the fraction of GFP⫹ cells which had become positive only in the previous 21 h (64⫾15% vs. 59⫾15%; P⫽0.8, n⫽6/group). There was a weak positive correlation (r⫽0.76; P⫽0.03) between the viability of a cell after NGF deprivation and the likelihood that it had become GFP⫹ during the 21 h of deprivation (Fig. 3).
DISCUSSION We used GFP as a marker of transfection in neurotrophindependent differentiated PC6-3 cells, a subline of the
Fig. 3. Boxes were etched on the bottoms of culture wells to create fields in which to observe cells. PC6-3 cells were grown in complete medium with 50 ng/ml nerve growth factor (NGF). On day 10, cells were transfected with pEGFP-C1 and the medium was replaced with serum-free medium containing NGF. On day 12, cells were deprived of NGF and immediately photographed under epifluorescence to document the transfection status of specific fields. Twenty-one hours later, cells were incubated with Hoechst 33258 and the same fields photographed under epifluorescence to determine the transfection status and viability of cells. The number of cells transfected in each field increased during this 21-h period, while the viability of the GFP⫹ cells continued to remain significantly high, even in the absence of NGF. These results are representative of two similar experiments.
PC12 cell line (Pittman et al., 1993). Transfection of NGF-deprived cells was associated with an apparent protection of GFP⫹ cells deprived of NGF. To determine whether this phenomenon was unique to GFP, -galactosidase was used as another marker of transfection, with similar results. This implies that it is not GFP which is neuroprotective, but the transfection process. The transfection reagent components were also tested, but none individually were able to block cell death after neurotrophin deprivation. Staurosporine (Bertrand et al., 1994) and H2O2 (Whittemore et al., 1994) were used to induce apoptosis by two other methods. No apparent protection effect was seen when transfected cells were treated with either staurosporine or H2O2, suggesting that the neuroprotection is relatively specific to apoptosis induced by neurotrophin deprivation. What could explain these observations? We considered three mechanisms, and designed experiments to distinguish between them (Fig. 4): (1) removing NGF from cells resistant to the effects of neurotrophin deprivation could cause increased transfection efficiency or expression of the transfected protein; (2) the process of transfection could cause cells to become resistant to neurotrophin deprivation; or (3) transfected cells expressing the target protein could become resistant to neurotrophin deprivation. Our results showed that the fraction of GFP⫹ cells which became positive over 21 h was the same in the NGF-deprived and NGF-maintained groups, and there was a positive correlation between the viability of NGF-deprived GFP⫺ cells and the likelihood that they would become GFP⫹ during the 21 h of deprivation. These findings
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Fig. 4. Three different mechanisms proposed to explain the increased survival of transiently transfected nerve growth factor-deprived neurons. In mechanism I, it is hypothesized that nerve growth factor (NGF) deprivation promotes increased efficiency of transfection or higher levels of expression of the transfected protein. If this were the case, then higher percentages of GFP⫹ cells would be seen in the NGF-deprived condition than the NGF-maintained condition. In mechanism II, the process of being transfected prevents the cell from NGF deprivation-induced death. This would imply that higher viability would be seen in conditions where there is more transition from GFP⫺ to GFP⫹. In mechanism III, the overexpressed protein itself protects from NGF deprivation–induced death. This would entail higher viability in cells that were already GFP⫹, compared with those that transition from GFP⫺ to GFP⫹.
are consistent with a model whereby cells that are transfected and about to express GFP are relatively protected from NGF deprivation (mechanism 2). If, instead, NGF deprivation induced GFP expression (mechanism 1), then there should have been increased numbers of cells becoming GFP⫹ in the NGF-deprived group, compared with the NGF-maintained group, contrary to what was found. If the GFP protein itself were protective from NGF deprivation (mechanism 3), then cells that were GFP⫹ at the beginning of the 21-h incubation period should have been more protected from NGF deprivation than cells that became GFP⫹ during the incubation period. This would have resulted in a negative correlation between the viability of the cell after NGF deprivation and the likelihood that a cell would change from GFP⫺ to GFP⫹, contrary to the positive correlation which was found. Although this study was limited to transient transfection of PC6-3 cells in NGF deprivation-induced apoptosis, we found the same rescue effect transfecting retinal ganglion cells in primary culture (Schmehil and Levin, unpublished observation), suggesting that these findings may apply to transfection of neuronal cells in general. While GFP and -galactosidase were the only two transfection marker proteins tested, they are structurally and functionally different, and therefore it is likely that the observed results apply to other markers. Similarly, the use of two different promoters (the CMV immediate early protein and the SV40 early promoter) makes it unlikely that there was an interaction between a specific trans-acting factor affecting transgene expression and the mechanism signaling apo-
ptosis after NGF deprivation (Wiebusch and Hagemeier, 1999). Transient transfection may therefore activate a neuroprotective response within the cell, similar to the protection of axotomized retinal ganglion cells from apoptosis by transduction of an adenoviral vector alone (Kugler et al., 1999). We studied neurotrophin deprivation-induced apoptosis, as this type of cell death is vital to the understanding of apoptosis in neuronal cells. The protective effect of transfection was only seen in this type of cell death, as induction of cell death with staurosporine and H2O2 was not protected by the transfection process. This implies that the transfection process does not induce a general survival response, but rather a neuroprotective response relatively specific to neurotrophin deprivation. It is therefore likely that transient transfection affects one of the pathways for survival downstream of tyrosine receptor kinase A occupancy, e.g. phosphatidylinositol 3-kinase, Akt, or NF-B activation, or c-Jun N-terminal kinase (JNK) inhibition (Fig. 5). The fact that transient transfection was not protective of H2O2-induced apoptosis suggests that the survival pathways activated by transfection are upstream of cytochrome c release and caspase activation (Kirkland and Franklin, 2001) and do not involve JNK inhibition (Maroney et al., 1999), while the failure of transient transfection to protect against staurosporine-induced apoptosis implies that the survival response is upstream of or parallel to phosphorylation of the Akt target p70 S6 kinase (Tee and Proud, 2001). A possible interaction between transfection and neuroprotection is related to the cell cycle (Jung and Fleming-
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Fig. 5. Some of the pathways responsible for neuronal survival in the presence of nerve growth factor and death when nerve growth factor is withdrawn (䊝). Transient transfection protects from nerve growth factor deprivation, implying that it either promotes one of the survival pathways or inhibits an apoptosis-inducing pathway. Transient transfection does not protect from the apoptosis induced by staurosporine (SS) and hydrogen peroxide (H2O2), implying that their targets are downstream of the neuroprotective effect. These have been grayed out. The dashed lines indicate multiple intermediate steps. Presumably one of the pathways depicted or alternative pathways are activated by transient transfection. There is a hypothetical relationship between cell survival and the effects of transient transfection on an abortive transition from G0 to G1; see discussion.
ton, 2001; Renzing and Lane, 1995; Rodriguez and Flemington, 1999). Calcium phosphate precipitate can cause growth arrest of dividing fibroblasts in the absence of plasmid DNA (Renzing and Lane, 1995). The presumption is that calcium phosphate crystals cause cellular stress. Calcium phosphate-mediated entry of DNA into the cell induces growth arrest in G0/G1, and this can induce apoptosis in a p53-independent manner (Jung and Flemington, 2001; Rodriguez and Flemington, 1999). There were similar findings in keratinocytes transfected with a liposomalbased method (Jensen et al., 2000), and it is possible that DNA entry associated with membrane disruption and the sealing process affects the cell cycle. Even though postmitotic neurons do not progress through the cell cycle, an abortive progression beyond the G1/S transition may be a necessary element for neuronal apoptosis induced by neurotrophin deprivation, excitotoxicity, and potassium deprivation (Farinelli and Greene, 1996; Frade, 2000; Freeman et al., 1994; Kranenburg et al., 1996; Liu and Greene, 2001; Martin-Romero et al., 2000; Pabmanabhan et al., 1999), but not serum deprivation (Lindenboim et al., 1995). If transient transfection arrested the neuron in G0/G1, then this could explain the prevention of apoptosis in differentiated PC6-3 cells undergoing NGF deprivation, similar to the inhibition of apoptosis when PC12 cells are arrested at G1/S (Farinelli and Greene, 1996), cyclin-dependent ki-
nases are inhibited (Park et al., 1996), or the E2F2 transcription factor is overexpressed (Persengiev et al., 2001). Future studies should clarify whether the transfectionassociated neuroprotection of neurotrophin deprivation results from interaction with an abortive progression through the cell cycle, activation of a known survival pathway, or a novel mechanism. Acknowledgements—Supported by National Institutes of Health R01 EY12492, the Glaucoma Foundation, the Retina Research Foundation, and an unrestricted departmental grant from Research to Prevent Blindness, Inc. LAL is a Research to Prevent Blindness Dolly Green Scholar.
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(Accepted 29 July 2002)