In the initial experiment, we utilized flow cytometric analysis to examine the localization of Fas to the cell surface of freshly isolated embryonic rat (gestational day (GD)-15) cortical neural cells by immuno-labeling for Fas in the absence of detergent. At this developmental age, the neuroepithelium proliferates rapidly and is the dominant cortical structure, and therefore, neuroepithelial precursors are the most abundant component of this tissue. Furthermore, at this age, the neuroepithelium mainly consists of neuronal (as opposed to glial) precursors (for review, see 40). Flow cytometric analysis of samples obtained from GD15 rat cortex indicated that 10.69 ± 3.37% (mean ± SEM) of cortical neuronal precursors expressed Fas on their cell surface in vivo on embryonic day 15 (data obtained from 6 independent samples of E15 cortex, Figure 1A). Once cortical precursors are cultured however, immunohistochemical analysis of detergent-permeabilized cultures (a measure of total Fas expression) indicates that a majority of these differentiating cells localize Fas-immunoreactivity within their cytoplasm and proximal processes (Figure 1B vs. control Figure 1C and 9). In contrast, flow cytometric analyses of non-permeabilized cultured embryonic cortical-derived neuronal precursors (Figure 2) indicate that 23–28% of these cells expressed Fas on their cell surface in vitro, representing ~2-fold induction of cell surface Fas expression in vitro, as compared to in vivo. Collectively, these data indicate that only a fraction (~1/4^th) of all Fas-expressing cells actually localize Fas to the cell-surface, in vitro.

The protein FADD (Fas-Associated Death Domain) is the major intracellular death-inducing signaling complex (DISC) adapter protein that couples Fas to intracellular caspase pathways 41424344, while FLIP is the major proximal intracellular protein that blocks Fas/Apo-1-mediated apoptosis 45. Since FADD and FLIP are antagonistic elements of the DISC-complex and are important intracellular gates of Fas/Apo-1 activation, we examined the extent to which these functionally divergent proteins co-localized with Fas/Apo-1 in differentiating embryonic cortical-derived neuroblasts. Two- parameter FACS analysis (Figure 1D) showed that, among adherent cultures, 72.5 ± 2.1% of Fas-expressing neuroblasts also co-localized FADD. In contrast, only 23.9 ± 3.2% Fas-expressing cells co-localized the anti-apoptotic protein FLIP suggesting that only a portion of Fas/Apo-1-expressing embryonic cortical-derived neuronal precursors may be protected from Fas-induced apoptosis.

Using flow cytometric analyses, we identified three distinct populations of neural cells in vitro; based on differences in the intensity of cell-surface Fas expression (Figure 2). Approximately 3% of cortical cells expressed Fas on their cell surface at a high intensity (Fas^Hi), while 20% of cultured neural cells expressed moderate levels of Fas on their cell surface (Fas^Mod). In contrast, 77% of cultured cortical neural cells do not express Fas on their cell-surface above background levels (Fas^-ve). Cultured cortical cells were counterstained with propidium iodide (PI) to measure cellular DNA content and to stage Fas-expressing cells into apoptosis (defined as cells with less than G0 DNA content), cell cycle arrest (G0/G1) or cell cycle (G2/S/M) phases (Figure 2A,2B,2C,2D). Our results indicate that the largest proportion of neural cells in the Fas^Hi population were apoptotic (Figure 2E). Few Fas^Hi cells were in G0/G1 or S/G2/M stages of cell cycle. In contrast, within the Fas^Mod population, the greatest percentage of Fas-expressing cells was observed in G0/G1 (Figure 2F).

The majority of Fas^Hi cells were apoptotic. We therefore examined the extent to which Fas expression was related to specific apoptosis-sensitive cell-biological processes including cell-matrix interactions, cell proliferation, and the activation of cell-cycle inhibitory proteins like p53.

The loss of adhesion to the extracellular matrix has been reported to induce apoptosis (a process referred to as anoikis), by activating caspases like caspase-2 46 that are down-stream of suicide receptors like Fas/Apo-1. Such cell-matrix interactions appear to be contextually relevant to the developing brain as well, since deletion of the Matrix Metalloproteinase (MMP)-9 gene (consequently suppressing type-4 collagen degradation) leads to delayed migration of granule cells, and decreased apoptosis in the developing cerebellum 47. Since post-mitotic neuroblasts migrate to their lamina-specific positions within the cortical plate using guidance cues that are dependent on the integrity of the extra-cellular matrix, we sought to determine if there was a relationship between cell-adhesion and Fas/Apo-1 expression. Non-adherent cells were harvested and analyzed separately from cells that were adherent to the culture dish. Among adherent cells (Figure 3A,3B), a majority of Fas-expressing neural cells belonged to the Fas^Mod group. In contrast, most Fas^Hi neural cells were non-adherent (Figure 3C,3D) and predominantly apoptotic.

Since primary cultured cells of the Fas^Hi group were predominantly non-adherent, we sought to determine if the induction of 'anoikis' could lead to an increase in Fas/Apo-1 expression. We therefore cultured embryonic day 15 cortical-derived neuroblasts on a collagen substrate (rat tail collagen, UBI) and treated adherent cultures with 50 U of Collagenase-A (MMP-1, microbial metalloendopeptidase, EC 3.4.24.3, Sigma) for 24 hours. Treatment of adherent neuronal precursors with collagenase-A led to a statistically significant, 2.5-fold increase in the number of cells that expressed Fas/Apo-1 on their cell surface (Figure 3E). Because of this relationship between 'anoikis' and Fas/Apo-1 expression, subsequent flow cytometric analyses focused on cells that were adherent to the extracellular matrix deposited onto the culture dish.

We had previously observed that Fas activation was followed by a transient increase in the uptake of 5-Bromo-2'-deoxyuridine (BrdU, 9) a marker of DNA replication. Though this increase in BrdU incorporation was not related to the induction of cell cycle, we hypothesized that the expression of Fas itself was related to cell proliferation and DNA synthesis. We therefore utilized three-parameter flow cytometric analyses to examine the relationship between cell-surface Fas expression and BrdU incorporation among neuronal precursors that were in cell cycle arrest (G0/G1), or actively in cell cycle (G2/S/M). Cells were first immuno-labeled for Fas, then detergent permeabilized and immuno-labeled for BrdU incorporation. Using propidium iodide (PI) fluorescence intensity as a classification parameter, cortical precursors were classified as belonging to one of three phases, G2/S/M, G0/G1, or apoptosis (sub-G0 DNA content). BrdU incorporation was significantly correlated with the cell-surface expression of Fas. In G0/G1 and S/G2/M conditions, correlation coefficients (r) ranged from 0.98 to 0.99, p < 0.05 (Figure 4A,4B). These data suggest that Fas/Apo-1 is most highly expressed in neuronal precursors that have recently completed their final cycle (for neuroblasts in G0/G1) or recently exited S-phase (for neuroblasts in S/G2/M). Furthermore, among cells in cell cycle (S/G2/M, Figure 4A), a discrete group of Fas^Hi precursors (arrows) also expressed the highest levels of BrdU incorporation. During cell cycle, the highest levels of BrdU incorporation are attained at the completion of chromosome replication (4N DNA content at the end of S-phase) and before the completion of mitosis. Therefore, G2 and early mitosis may represent periods during which some sub-populations of proliferating cortical neuroblasts are particularly vulnerable to receptor-mediated suicide signals. Our data therefore indicate that while Fas activation does not induce or suppress cell cycle 9, the highest expression of Fas is associated with progression past the S-phase of cell cycle or recent exit from cell cycle.

The transcription factor p53 induces Fas gene transcription 33, the translocation of Fas from Golgi complex to cell surface 34, and activates Fas-mediated apoptosis in un-transformed vascular smooth muscle cells 34 and mammary epithelial tumors 35. We had previously shown that p53 activation in cortical progenitor cells was associated with the induction of the mitochondrial death-associated protein Bax 37. We therefore examined the extent to which p53 status predicted the expression of Fas/Apo-1 on the cell surface of cortical neuronal precursors. We used phosphorylation of p53 at serine 392 as a marker for p53 activation 37. Cells were first immuno-labeled for Fas, then detergent-permeabilized and immuno-labeled for p(phospho)p53. The intensity of pp53 expression was statistically significantly correlated (p < 0.05, r = 0.73, Figure 5A) with the intensity of cell surface Fas expression in primary cortical neuronal precursors. Thus, cells that exhibit high levels of p53 phosphorylation also express high cell-surface levels of Fas.

As a follow-up experiment, to determine whether p53 activation is required for the expression of Fas/Apo-1, we utilized a rodent cerebral cortical cell line (CHB50), conditionally immortalized with a temperature sensitive mutation of the SV40 large T antigen (tsTA), that we had previously developed and characterized 37. In the +tsTA condition (incubation at 33°C), neuroblasts proliferate indefinitely and p53 is inactive as indicated by the absence of phosphorylated p53 (-pp53). However, cessation of large T antigen expression (-tsTA, 39°C) is accompanied by p53 phosphorylation (+pp53) and the induction of p53-dependent proteins like Bax 37. CHB50 neuroblasts were therefore cultured under +tsTA and -tsTA conditions. Western immunoblot analysis indicated that CHB50 neuroblasts expressed very low-to-undetectable levels of Fas in the -pp53 (+tsTA) condition. However, following the activation of p53, [+pp53 (-tsTA) condition], there was a statistically significant 12-fold increase in the expression of Fas, 48 hours following activation of p53 (Figure 5B,5C).

We had previously observed that estrogen prevented apoptosis following p53 activation and decreased the expression of the mitochondrial-associated pro-apoptotic protein Bax in differentiating cerebral cortical neuroblasts 37. Furthermore, in the context of p53 activation, estrogen led to an increase in neuroblast proliferation. We therefore examined the extent to which cell-surface Fas expression was estrogen-dependent.

Flow cytometric analysis of non-synchronized GD15 cortical neuronal precursors (Figure 6A) indicated that 24 hours exposure to estradiol-17β (E2) at 2 nM led to a statistically significant decrease in the total number of cortical cells in G0 (Figure 6B) and a corresponding significant increase in the number of neuroblasts in the DNA synthesis (S)-phase (Figure 6C). At 24 hours there was not a statistically significant change in the numbers of cells progressing through G2/M (Figure 6D), suggesting that estrogen-stimulated cells may have not yet completed cell cycle. There was also a non-statistically-significant trend towards a decrease in the total number of neuronal precursors that were apoptotic (Figure 6E). Our previous data also showed that estrogen decreased the expression of another member of the Fas family, the pan-neurotrophin receptor p75^NTR 4849. We therefore also examined estrogen regulation of cell-surface Fas expression at different stages of cell cycle.

As observed earlier, in adherent embryonic cortical cultures, most Fas expressing cells belong to the Fas^Mod category and very few Fas^Hi-type cells can be observed (Figure 7A, control). Twenty-four hours of exposure to 2 nM E2 led to a statistically significant, 1.5-fold decrease in the percentage of cells in G0/G1 that expressed Fas (Figure 7A:E2 and 7B). In contrast, the numbers of Fas expressing cells in cell cycle, S/G2/M, were not altered by estrogen exposure (Figure 7C), though estrogen led to a statistically significant 2-fold decrease in the intensity of cell-surface Fas expression (on a per-cell basis, Figure 7D). Additionally, estrogen led to a statistically significant, 2-fold decrease in the numbers of Fas-expressing cells that were apoptotic, suggesting that estrogen suppresses apoptosis in Fas-expressing cells (Figure 7E).

Since Fas expression was also associated with p53 activation, we hypothesized that estrogen would suppress Fas expression in the context of p53 activation as well. We therefore examined the expression of Fas in tsTA-CHB50 cortical neuroblasts cultured for 24 and 72 hours under +pp53 (-tsTA) conditions, and treated for those durations with either estradiol-17β (2 nM) alone or estradiol-17β with the antagonist 4-hydroxytamoxifen (at 1 uM, Sigma). Our previous western immunoblot analyses indicated that in control CHB50 cortical cultures, Fas expression was statistically significantly induced 72 hours following p53 activation (Figure 5B,5C). In the context of p53 activation, estrogen led to a significant decrease in the expression of Fas at 72 hours but not at 24 hours (Figure 8). Surprisingly (see discussion), concurrent exposure to the estrogenic antagonist 4-hydroxytamoxifen did not prevent the estrogen-induced reduction in Fas.

Flow cytometric analysis of primary cortical cultures indicated that estrogen led to an increase in the number of cells in S-phase (Figure 6A,6C), consistent with our previously published observations that estrogen promotes neurogenesis in cortical neuroblasts 37. Furthermore, estrogen prevented apoptosis in cortical neuroblasts. We therefore hypothesized that in the context of Fas activation, estrogen would alter the expression of specific cell-cycle related proteins such as Cyclin-A 50, the Proliferating Cell Nuclear Antigen (PCNA, 5152) and p21/waf-1 53, that also serve anti-apoptotic and DNA repair functions in addition to their roles in cell cycle. For example, PCNA and p21/Waf-1 bind to each other to prevent apoptosis induction 51, and to promote repair of double strand breaks in DNA, a characteristic of apoptosis. We therefore examined if Fas activation and estrogen could interact to regulate the expression of cyclin A, PCNA and p21/waf-1 and compared the expression of these anti-apoptotic cell cycle proteins with other cell-cycle proteins. Dissociated embryonic cortical cells were exposed to soluble Fas ligand (sFasL, 5 ng/ml, Alexius Corp) or estradiol-17β (2 nM) either alone or concurrently, for 12 hours. Western immunoblot analysis indicated that neither estrogen nor FasL alone led to a change in the expression of PCNA. However, concurrent exposure to both estrogen and FasL did lead to a significant decrease in PCNA expression (Figure 9). Since p21/Waf-1 may prevent apoptosis by interacting with PCNA 51, we examined the expression of p21/Waf-1 in dissociated embryonic cortical cells that were exposed to sFasL, estrogen or both sFasL and estrogen for 12 hours. Neither estrogen, nor sFasL alone induced an alteration in p21/Waf1 expression, though sFasL-treated cultures exhibited a non-statistically significant trend towards decreased p21/waf-1 expression. However, the concurrent administration of estrogen and sFasL led to a significant decrease in the expression of p21/Waf-1 (Figure 9).

Cyclin-A prevents apoptosis by disrupting the formation of a complex between the E2F1 and p53 transcription factors 50. Therefore, we examined the expression of cyclin-A following exposure to E2 and/or FasL. Both estrogen and sFasL alone led to a statistically significant increase in the expression of cyclin-A compared with controls at 12 hours post-treatment. Concurrent exposure to sFasL and estrogen was not different from exposure to either agent alone (Figure 9).

Neither estradiol-17β nor sFasL alone or together altered the expression of cdk2 or cyclin E compared to control cultures (Figure 9). Similarly, neither estrogen nor sFasL significantly altered the expression of Cyclin-dependent Kinase-1 (cdk1/cdc2) relative to control cultures, suggesting that that interactions between estrogen and Fas activation were not specifically related to cell cycle.

Timed-pregnant rats (Sprague Dawley) were purchased from Harlan (TX). Gestational Day (GD) zero was defined as the day on which dams were sperm positive. GD-15 rat fetuses were obtained under aseptic conditions, from pregnant dams that were anesthetized with Phenobarbital (50 mg). Fetal brains were dissected out aseptically and, in all cases, care was taken to minimize any pain and discomfort to the animals.

The cerebral cortical mantle of GD-15 rat fetuses was dissected out under sterile conditions, and separated from overlying meningeal tissue under a microscope. We selected GD15 because this age represents the peak period for proliferation within the cortical neuroepithelium, to generate neurons of the cortical plate. At GD15, the neuroepithelium is the dominant cortical structure, though a smaller number of differentiated neurons (the cortical preplate) and radial glia are also present. Furthermore, at this age, the neuroepithelium is mainly comprised of proliferating neuronal (rather than glial) precursors (for review see 40). The earliest developing layer of the cortical plate (layer VI) does not form until one day later (GD16-17). Morphologically, these cortical cultures are immature and continue to proliferate in vitro, but over a period of 24 hours, cultured neuroblasts become increasingly polarized and exhibit growth cones and process characteristic of early differentiating neurons. Cultures were established according to our previously published protocols 9. Briefly, neuroblasts were dispersed by trituration in 0.5% trypsin and 6.84 mM EDTA (Sigma) and were plated on collagen-coated 24-well plates in sterile culture medium (89% Dulbecco's modified eagle media [DMEM], 10% gelding serum and 1% penicillin-streptomycin). Cultures were maintained at 37°C for 24 hours and the culture medium was replaced. For initial experiments, whole cultures were assayed 24 hours later by FACS (Fluorescence-assisted cell sorting) analysis. For subsequent experiments, only adherent cells were harvested at 24 hours and fixed for FACS analysis. For some experiments, soluble protein was extracted for western immunoblot analysis. Some experimental groups were exposed to soluble Fas Ligand (sFasL 5 ng/ml, Alexis Corp.), estradiol-17β (2 nM, Sigma) or sFasL together with estradiol-17β. Control and sFasL-treated cultures were maintained at 37°C in normal culture medium for 24 hours and then exposed to the appropriate treatments. Estrogen-treated cultures were pre-treated with estradiol-17β for 24 hours and then re-fed with estrogen alone or estrogen concurrently with sFasL for 12 hours.

To examine the regulation of Fas by p53 and estrogen, we also utilized a rodent cerebral cortical cell line (CHB50) conditionally immortalized with a temperature sensitive mutation of the SV40 large T antigen (tsTA) that we had previously developed and characterized 37. In the +tsTA condition (incubation at 33°C), neuroblasts proliferate indefinitely and p53 is inactive. However, cessation of large T antigen expression (-tsTA, 39°C) is accompanied by induction of the p53 phosphorylation and the induction of p53-dependent proteins, p21/Waf1, Bax and MDM2 that lead to cell cycle-arrest, suicide, and p53 inhibition/cell cycle re-entry respectively. We had previously observed that CHB50 neuroblasts express the estrogen receptor ER-α and are estrogen-responsive. Estrogen prevents apoptosis following p53 activation and induces both neurogenesis and neuronal differentiation in this cell line. CHB50 cortical neuroblasts were therefore cultured under the +tsTA and -tsTA conditions as published previously 37 to examine the p53-related expression of Fas, and under the -tsTA conditions to examine the estrogen regulation of Fas during neuroblast differentiation (in the context of p53 activation).

Triple parameter flow cytometric analysis was used to measure immunolabeled cell surface Fas protein, BrdU incorporation (a measure of DNA incorporation) and DNA content [propidium iodide (PI) dye intercalation] as a measure of cell cycle distribution. Dissociated embryonic cortical cultures were administered a single BrdU pulse (24 hours) at the time the cultures were established. Non-adherent and adherent [harvested by administration of collagenase (Sigma, 30 units)] cells were obtained after the initial 24-hour BrdU pulse and centrifuged at 300 × g to sediment the cells. The pellet was washed with Dulbecco's Phosphate buffered saline (PBS) and fixed in 1% paraformaldehyde and re-suspended in PBS. Cells were sedimented by centrifugation between subsequent immunohistochemical steps. To detect cell-surface Fas/Apo-1, immuno-histofluorescence chemistry was performed in the absence of detergent. Following exposure to a blocking solution (in Tris-buffered saline, (TBS) pH 7.4, with 0.1%Bovine Serum Albumin, and 2% Normal Goat Serum), cell-surface Fas/Apo-1 expression was detected with a rabbit polyclonal anti-Fas antibody (Santa Cruz, 1:500), followed by biotinylated, secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. Following Fas/Apo-1 immunofluorescence, cells were permeabilized with detergent (0.3% Trition-X100) in blocking solution. BrdU incorporation was detected with a primary anti-BrdU antibody (Sigma, 1:500) followed by biotinylated, secondary horse anti-mouse (Vector, 1:1000), conjugated to streptavidin-allophycocyanin (APC, Molecular Probes, 1:500). The cells were then washed with PBS and exposed to a PI solution (see above) for 10 minutes at room temperature. BrdU-allophycocyanin, Fas-FITC and PI fluorescence was analyzed from 10⁴ cells with excitation at 488 nm (argon laser) and detection of FITC fluorescence at approximately 530 nm, PI fluorescence at 585 nm and allophycocyanin at 650 nm.

In studies examining the relationship between cell surface Fas and Fas-associated protein expression, binding of the primary antibody, mouse monoclonal antibody to Fas (Transduction Laboratories, 1:500), was detected using a rat-absorbed, biotinylated horse-anti-mouse secondary antibody (Vector, 1:1000), conjugated to streptavidin-APC or rabbit polyclonal anti-Fas antibody (Santa Cruz, 1:500), followed by biotinylated, secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. Subsequently, FLIP or phosphorylated P53 was detected using rabbit polyclonal anti-FLIP (gift from Drs. Sato and Walsh, Tufts University School of Medicine; 1:500) or phospho-p53 at serine 392 (New England BioLabs, 1:500) followed by a secondary donkey anti-rabbit (Jackson Immunochemicals, 1:1000) conjugated to streptavidin-FITC. FADD was detected using a monoclonal anti-FADD antibody (Transduction Laboratories, 1:500) followed by a rat-absorbed, biotinylated horse-anti-mouse secondary antibody (Vector, 1:1000). Phosphorylation of the casein kinase II-sensitive site at serine 392 was used as a marker for activated p53. Phosphorylation at this site promotes tetramerization of p53 90 and specificity of p53 binding to DNA 91, while regulating re-annealing of double-stranded DNA 92. Cells were then washed with PBS and exposed to a PI solution [PBS, 0.1% triton (Sigma), 0.5 mmol EDTA, 0.05 mg/ml RNAse A and 50 mg/ml PI] for 10 minutes at room temperature. Fas-FITC and PI fluorescence was analyzed from 10,000 cells using a flow cytometer (Becton Dickinson) with excitation at 488 nm (argon laser) and detection of FITC fluorescence at approximately 530 nm and PI fluorescence at 585 nm. The specificity of all antibodies used for flow cytometric analysis was previously verified by western immunoblot analysis and/or immunohistochemical analysis 937. Immunological controls were exposed to pre-immune serum in place of the appropriate primary antibody and were used to determine background immunofluorescence.

The expression of cell cycle elements that regulate S-phase [proliferating cell nuclear antigen (PCNA)], the G1/S transition [cyclin dependent kinase 2 (cdk2) and cyclin E], the G2/M transition [cyclin dependent kinase (cdk1) and cyclin A], and a cell cycle arrest factor [p21/wild-type p53-activated fragment (Waf1)/Cip1 (cdk-interacting protein-1)], and Fas were verified by western immunoblot analysis of E15 cortical neuroblasts, according to our previously published protocols 9379394. Detergent (1% SDS) soluble protein was isolated using the Trizol reagent (Invitrogen). Protein samples were size fractionated on an 8% SDS-polyacrylamide gel, and blotted onto supported nitrocellulose (Hibond-C-super, Amersham). Blots were blocked (5% milk, TBS (1.4 M NaCl, 0.2 M Tris)-0.1% Tween 20), exposed to primary antibody [mouse monoclonal anti-PCNA antibody (Calbiochem, 1:66); rabbit polyclonal anti-cyclin E (Calbiochem, 1:250); mouse monoclonal anti-cyclin A (Calbiochem, 1:100), mouse monoclonal anti-cdk2 (Transduction Laboratories, 1:500), mouse monoclonal anti-cdk1 (Calbiochem, 1:100) or mouse monoclonal anti-Fas (Transduction Labs)], washed and exposed to horse-anti-mouse secondary antibody (Vector, 1:1000) or donkey anti-rabbit (Jackson Laboratories, 1:5000), washed again and exposed to streptavidin horseradish-peroxidase conjugate (Amersham). Immunoreactive bands were detected using enzyme-linked chemiluminescense (NEN).

Data analyses for the experiments reported here were based on 5–9 independent samples. For FACS analysis, the incorporation of PI into cellular DNA was used to categorize cells in G0/G1 (diploid DNA content), S/G2/M (hyper-diploid DNA content) and apoptosis (sub-diploid DNA content) 959697. Cells were categorized, and intensities calculated using standard cell sorting software (Cell Quest, Becton Dickinson). Western immunoblots were analyzed using standard densitometric software (Molecular Analyst, Bio-Rad). Sample size for western blot analyses was 6. Statistical differences were calculated using ANOVAs followed by post-hoc tests (Student-Newman-Keuls, p < 0.05). Correlational analyses were calculated by combining mean intensity values from 6–8 replications using Pearson's product moment correlation (r) followed by a two-tailed test for the significance of the correlation coefficient. Levels of statistical significance were set at p < 0.05. Data were expressed in terms of mean ± standard error of the mean (SEM).