Background The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E 2 -BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E 2 -BSA does not compete for E 2  binding to purified ER  in vitro . To resolve this apparent discrepancy, we performed competition studies examining the binding of E 2  and E 2 -BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E 2 -BSA preparations with unconjugated E 2  (usually between 3–5%), the latter was carefully removed by ultrafiltration.
Background
The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E 2 -BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E 2 -BSA does not compete for E 2  binding to purified ER  in vitro . To resolve this apparent discrepancy, we performed competition studies examining the binding of E 2  and E 2 -BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E 2 -BSA preparations with unconjugated E 2  (usually between 3–5%), the latter was carefully removed by ultrafiltration.in vitroResults As previously reported, a 10-to 1000-fold molar excess of E 2 -BSA was unable to compete with  3 H-E 2  binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E 2 -BSA, the binding of  3 H-E 2  was significantly reduced. E 2 -BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E 2 -BSA (E 2 -BSA-FITC). Staining was restricted to the cell membrane when E 2 -BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E 2  in a dose dependent manner and with the competitor ICI-182,780.
Results
As previously reported, a 10-to 1000-fold molar excess of E 2 -BSA was unable to compete with  3 H-E 2  binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E 2 -BSA, the binding of  3 H-E 2  was significantly reduced. E 2 -BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E 2 -BSA (E 2 -BSA-FITC). Staining was restricted to the cell membrane when E 2 -BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E 2  in a dose dependent manner and with the competitor ICI-182,780.Conclusions These results demonstrate that E 2 -BSA does bind to purified ER  in vitro  and to ER in intact cells. It seems likely that the size and structure of E 2 -BSA requires more energy for it to bind to the ER and consequently binds more slowly than E 2 . More importantly, these findings demonstrate that in intact cells that express ER, E 2 -BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.
Conclusions
These results demonstrate that E 2 -BSA does bind to purified ER  in vitro  and to ER in intact cells. It seems likely that the size and structure of E 2 -BSA requires more energy for it to bind to the ER and consequently binds more slowly than E 2 . More importantly, these findings demonstrate that in intact cells that express ER, E 2 -BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.in vitro

Background For many years, estrogen actions have been presumed to be mediated almost exclusively through the regulation of target gene transcription by a chromosomal bound estrogen receptor. These genomic estrogen effects are the well described interactions between the estrogen receptor and adapter transcription factors that result in activation or inhibition of the basal transcription protein machinery. However, there is a growing body of evidence that several rapid estrogen effects are non-transcriptional in nature. These rapid estrogen effects include changes of calcium flux in several cell types [ 1 - 3 ], MAPK activation [ 4 , 5 ], cAMP levels [ 6 , 7 ], and nitric oxide release [ 8 ]. That many of these effects are mediated by a membrane-localized estrogen receptor has been postulated for some time [ 9 , 10 ], but the majority of evidence supporting this hypothesis is indirect, relying on the induction of these non-genomic effects using estrogen covalently conjugated to BSA by a 6 atom hydrocarbon tether (E 2 -BSA) [ 11 , 12 ]. However, the relative binding efficiency of these conjugates is low and concern has been raised regarding the use of these conjugates as direct surrogates for estrogen [ 13 ]. A recent report added to this controversy by showing that commercially available E 2 -BSA is contaminated by unconjugated free E 2  and a series of binding experiments demonstrated that E 2 -BSA was unable to bind to ER after the contaminant E 2  was removed. [ 14 ]. These findings contradict studies where fluorescein-labeled E 2 -BSA (E 2 -BSA-FITC) specifically bound to a putative ER on the cell membrane [ 15 - 17 ]. Elucidation of novel membrane-associated ER effects is crucial to our understanding of the non-genomic signaling pathways of ER and other hormone receptors. Hormone-conjugated BSA is an important tool in this pursuit. We believe the contradictory results are explained by differences in the rates of binding of the bulky E 2 -BSA and E 2  with the ER. We show that pre-incubation of E 2 -BSA with ERα results in a highly significant decrease in the binding of  3 H-E 2 . The binding of  3 H-E 2  with ERα is unaffected by the simultaneous addition of E 2 -BSA. We also demonstrate that fluorescein conjugated E 2 -BSA binds to the membrane of cells that endogenously produce ERα and to HeLa cell lines stably expressing mERα.
Background
For many years, estrogen actions have been presumed to be mediated almost exclusively through the regulation of target gene transcription by a chromosomal bound estrogen receptor. These genomic estrogen effects are the well described interactions between the estrogen receptor and adapter transcription factors that result in activation or inhibition of the basal transcription protein machinery. However, there is a growing body of evidence that several rapid estrogen effects are non-transcriptional in nature. These rapid estrogen effects include changes of calcium flux in several cell types [ 1 - 3 ], MAPK activation [ 4 , 5 ], cAMP levels [ 6 , 7 ], and nitric oxide release [ 8 ]. That many of these effects are mediated by a membrane-localized estrogen receptor has been postulated for some time [ 9 , 10 ], but the majority of evidence supporting this hypothesis is indirect, relying on the induction of these non-genomic effects using estrogen covalently conjugated to BSA by a 6 atom hydrocarbon tether (E 2 -BSA) [ 11 , 12 ]. However, the relative binding efficiency of these conjugates is low and concern has been raised regarding the use of these conjugates as direct surrogates for estrogen [ 13 ]. A recent report added to this controversy by showing that commercially available E 2 -BSA is contaminated by unconjugated free E 2  and a series of binding experiments demonstrated that E 2 -BSA was unable to bind to ER after the contaminant E 2  was removed. [ 14 ]. These findings contradict studies where fluorescein-labeled E 2 -BSA (E 2 -BSA-FITC) specifically bound to a putative ER on the cell membrane [ 15 - 17 ].Elucidation of novel membrane-associated ER effects is crucial to our understanding of the non-genomic signaling pathways of ER and other hormone receptors. Hormone-conjugated BSA is an important tool in this pursuit. We believe the contradictory results are explained by differences in the rates of binding of the bulky E 2 -BSA and E 2  with the ER. We show that pre-incubation of E 2 -BSA with ERα results in a highly significant decrease in the binding of  3 H-E 2 . The binding of  3 H-E 2  with ERα is unaffected by the simultaneous addition of E 2 -BSA. We also demonstrate that fluorescein conjugated E 2 -BSA binds to the membrane of cells that endogenously produce ERα and to HeLa cell lines stably expressing mERα.Results E 2 -BSA binding to purified estrogen receptor Although E 2  is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E 2 -BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E 2 -BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E 2 -BSA, maximizing the amount of E 2 -BSA bound to the receptor. To test this hypothesis, E 2 -BSA free of contaminant E 2  was prepared by ultrafiltration. Competition between the purified E 2 -BSA and labeled E 2  for binding to purified ERα was determined after E 2 -BSA was pre-incubated with ERα and also when added at the same time as labeled E 2 . As shown in figure  1 , concurrent addition of labeled E 2  and E 2 -BSA had no effect on labeled E 2  binding. However, a four-hour pre-incubation of E 2 -BSA with ER significantly decreased E 2  binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E 2 -BSA. Figure 1 Pre-incubation of purified hERα with E 2 -BSA competes for estradiol binding.  Purified ERα was incubated with E 2  (solid line) or E 2 -BSA (dotted line) for four hours before (a) or concurrently with (b) the addition of labeled E 2 . Incubation was continued for another 2 h at room temperature, and at the end of this period, specific binding was determined by adsorption, removal, and counting of free labeled E 2 . E 2 -BSA binding to ER in intact cells Non-genomic actions of the estrogen receptor are now well established. Several investigators have demonstrated that fluorescein labeled E 2 -BSA (E 2 -BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E 2 . Specific binding of E 2 -BSA-FITC to this membrane-localized form of the ER would further establish that E 2  conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E 2 -BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E 2  to HeLa-ERα, but not native HeLa cells (figure  2 ). Scatchard analysis of the binding of E 2  to HeLa-ERα cells showed that although weakly expressed, the Kd for the expressed ERα was 7.04 nM, similar to published values (figure  3 ). HeLa-ERα cells, but not native HeLa cells, exhibited fluorescent staining of the cell membrane after incubation with E 2 -BSA-FITC (figure  4 ). The heterogeneous staining pattern reflected the low level of ERα expression. This fluorescence was not seen when HeLa-ERα cells were incubated with BSA conjugated to fluorescein alone (data not shown). Figure 2 Whole cell binding of estradiol to MC7 cells and HeLa cells stably transfected with ERα.  2 × 10 6  cells were incubated with  3 H-17β-estradiol (10 -8  M) in the absence (solid) and presence (white) of 100 fold excess of unlabeled estradiol for 15 minutes at room temperature, washed, and placed on ice for 30 minutes. Cells were then pelleted, lysed and counted. Results are expressed as the mean +/- the SEM of 3 experiments (* p < 0.01) Figure 3 Estradiol binding to HeLa cells stably transfected with ERα.  Subconfluent HeLa-ERα cells were trypsinized and aliquots (2 × 10 6  cells) incubated with several concentrations of  3 H-17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. a) Representative binding results of 3 independent experiments with total binding (solid box), non-specific binding (open box), and specific binding (triangle). b) Scatchard analysis of binding results. Figure 4 Membrane localization of ERα.  HeLa cells stably transfected with ERα(a) and native HeLa cells (b) were incubated with fluorescein labeled membrane impermeable BSA conjugated to estradiol (E 2 -BSA-FITC) and visualized under phase contrast bright field and with UV light with an excitation filter for FITC. To establish the specificity of E 2 -BSA binding, MC7 cells and HeLa-ERα cells were incubated with E 2 -BSA-FITC after pre-incubation with various concentrations of E 2  and the anti-estrogen ICI-182,780. As shown in figure  5 , fluorescence was lost in both cell types in a dose dependent manner with increasing concentrations of E 2 . Fluorescence was almost completely eliminated by pre-incubation with the specific competitor ICI-182,780. BSA conjugated to FITC alone did not bind. These results suggest that estrogen covalently bound to BSA can bind to ER in a biologically significant manner. Figure 5 E 2  competition with E 2 -BSA-FITC binding.  MC7 cells (black bars) or HeLa-ERα (white bars) were incubated with vehicle, various concentrations of E 2 , or ICI 182,780 (10 -8  M) for 30 minutes and then incubated an additional 30 minutes with E 2 -BSA-FITC or BSA-FITC alone (grey bar). Cells were fixed, and visualized by confocal microscopy. Digitized images were inverted to black on white and pixel density for each cell was determined by averaging the density across the cell membrane at four orthogonal points. Each bar represents >20 cells counted +/- SEM. (* p < 0.05). The possibility that E 2 -BSA-FITC could be degraded during incubation with intact cells was examined using HPLC. E 2 -BSA-FITC was incubated in empty wells or wells containing MC7 cells under the same conditions employed for the binding studies described above. Media was removed from the cells and HPLC performed with a reverse phase column. Peaks were visualized using a scanning fluorescence detector. Aqueous solutions of E 2 -BSA-FITC produced a single peak with a retention time of 5.5 minutes using a methanol-water gradient from 80% to 50% over 30 minutes at 1 ml/minute. E 2  and E 2 -BSA did not fluoresce at the excitation and emission wavelengths used (data not shown). Spectra obtained from media containing E 2 -BSA-FITC alone and media containing E 2 -BSA-FITC incubated with MC7 cells are shown in figure  6 . The average area under the curve for E 2 -BSA-FITC was the same (p < 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E 2 -BSA-FITC is stable under the culture conditions employed for the binding experiments. Figure 6 Stability of E 2 -BSA-FITC.  E 2 -BSA-FITC (10 -8  M in estrogen) was placed in empty wells or wells containing MC7 cells and incubated for 30 minutes at 4°C. E 2 -BSA-FITC was detected by reverse phase HPLC using a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at 1.0 ml/min. The assay was run in triplicate. Representative spectra are shown for E 2 -BSA-FITC alone (A) and E 2 -BSA-FITC incubated with MC7 cells (B).
Results
E 2 -BSA binding to purified estrogen receptor Although E 2  is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E 2 -BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E 2 -BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E 2 -BSA, maximizing the amount of E 2 -BSA bound to the receptor. To test this hypothesis, E 2 -BSA free of contaminant E 2  was prepared by ultrafiltration. Competition between the purified E 2 -BSA and labeled E 2  for binding to purified ERα was determined after E 2 -BSA was pre-incubated with ERα and also when added at the same time as labeled E 2 . As shown in figure  1 , concurrent addition of labeled E 2  and E 2 -BSA had no effect on labeled E 2  binding. However, a four-hour pre-incubation of E 2 -BSA with ER significantly decreased E 2  binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E 2 -BSA. Figure 1 Pre-incubation of purified hERα with E 2 -BSA competes for estradiol binding.  Purified ERα was incubated with E 2  (solid line) or E 2 -BSA (dotted line) for four hours before (a) or concurrently with (b) the addition of labeled E 2 . Incubation was continued for another 2 h at room temperature, and at the end of this period, specific binding was determined by adsorption, removal, and counting of free labeled E 2 .
E 2 -BSA binding to purified estrogen receptor
Although E 2  is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E 2 -BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E 2 -BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E 2 -BSA, maximizing the amount of E 2 -BSA bound to the receptor. To test this hypothesis, E 2 -BSA free of contaminant E 2  was prepared by ultrafiltration. Competition between the purified E 2 -BSA and labeled E 2  for binding to purified ERα was determined after E 2 -BSA was pre-incubated with ERα and also when added at the same time as labeled E 2 . As shown in figure  1 , concurrent addition of labeled E 2  and E 2 -BSA had no effect on labeled E 2  binding. However, a four-hour pre-incubation of E 2 -BSA with ER significantly decreased E 2  binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E 2 -BSA.E 2 -BSA binding to ER in intact cells Non-genomic actions of the estrogen receptor are now well established. Several investigators have demonstrated that fluorescein labeled E 2 -BSA (E 2 -BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E 2 . Specific binding of E 2 -BSA-FITC to this membrane-localized form of the ER would further establish that E 2  conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E 2 -BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E 2  to HeLa-ERα, but not native HeLa cells (figure  2 ). Scatchard analysis of the binding of E 2  to HeLa-ERα cells showed that although weakly expressed, the Kd for the expressed ERα was 7.04 nM, similar to published values (figure  3 ). HeLa-ERα cells, but not native HeLa cells, exhibited fluorescent staining of the cell membrane after incubation with E 2 -BSA-FITC (figure  4 ). The heterogeneous staining pattern reflected the low level of ERα expression. This fluorescence was not seen when HeLa-ERα cells were incubated with BSA conjugated to fluorescein alone (data not shown). Figure 2 Whole cell binding of estradiol to MC7 cells and HeLa cells stably transfected with ERα.  2 × 10 6  cells were incubated with  3 H-17β-estradiol (10 -8  M) in the absence (solid) and presence (white) of 100 fold excess of unlabeled estradiol for 15 minutes at room temperature, washed, and placed on ice for 30 minutes. Cells were then pelleted, lysed and counted. Results are expressed as the mean +/- the SEM of 3 experiments (* p < 0.01) Figure 3 Estradiol binding to HeLa cells stably transfected with ERα.  Subconfluent HeLa-ERα cells were trypsinized and aliquots (2 × 10 6  cells) incubated with several concentrations of  3 H-17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. a) Representative binding results of 3 independent experiments with total binding (solid box), non-specific binding (open box), and specific binding (triangle). b) Scatchard analysis of binding results. Figure 4 Membrane localization of ERα.  HeLa cells stably transfected with ERα(a) and native HeLa cells (b) were incubated with fluorescein labeled membrane impermeable BSA conjugated to estradiol (E 2 -BSA-FITC) and visualized under phase contrast bright field and with UV light with an excitation filter for FITC. To establish the specificity of E 2 -BSA binding, MC7 cells and HeLa-ERα cells were incubated with E 2 -BSA-FITC after pre-incubation with various concentrations of E 2  and the anti-estrogen ICI-182,780. As shown in figure  5 , fluorescence was lost in both cell types in a dose dependent manner with increasing concentrations of E 2 . Fluorescence was almost completely eliminated by pre-incubation with the specific competitor ICI-182,780. BSA conjugated to FITC alone did not bind. These results suggest that estrogen covalently bound to BSA can bind to ER in a biologically significant manner. Figure 5 E 2  competition with E 2 -BSA-FITC binding.  MC7 cells (black bars) or HeLa-ERα (white bars) were incubated with vehicle, various concentrations of E 2 , or ICI 182,780 (10 -8  M) for 30 minutes and then incubated an additional 30 minutes with E 2 -BSA-FITC or BSA-FITC alone (grey bar). Cells were fixed, and visualized by confocal microscopy. Digitized images were inverted to black on white and pixel density for each cell was determined by averaging the density across the cell membrane at four orthogonal points. Each bar represents >20 cells counted +/- SEM. (* p < 0.05). The possibility that E 2 -BSA-FITC could be degraded during incubation with intact cells was examined using HPLC. E 2 -BSA-FITC was incubated in empty wells or wells containing MC7 cells under the same conditions employed for the binding studies described above. Media was removed from the cells and HPLC performed with a reverse phase column. Peaks were visualized using a scanning fluorescence detector. Aqueous solutions of E 2 -BSA-FITC produced a single peak with a retention time of 5.5 minutes using a methanol-water gradient from 80% to 50% over 30 minutes at 1 ml/minute. E 2  and E 2 -BSA did not fluoresce at the excitation and emission wavelengths used (data not shown). Spectra obtained from media containing E 2 -BSA-FITC alone and media containing E 2 -BSA-FITC incubated with MC7 cells are shown in figure  6 . The average area under the curve for E 2 -BSA-FITC was the same (p < 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E 2 -BSA-FITC is stable under the culture conditions employed for the binding experiments. Figure 6 Stability of E 2 -BSA-FITC.  E 2 -BSA-FITC (10 -8  M in estrogen) was placed in empty wells or wells containing MC7 cells and incubated for 30 minutes at 4°C. E 2 -BSA-FITC was detected by reverse phase HPLC using a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at 1.0 ml/min. The assay was run in triplicate. Representative spectra are shown for E 2 -BSA-FITC alone (A) and E 2 -BSA-FITC incubated with MC7 cells (B).
E 2 -BSA binding to ER in intact cells
Non-genomic actions of the estrogen receptor are now well established. Several investigators have demonstrated that fluorescein labeled E 2 -BSA (E 2 -BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E 2 . Specific binding of E 2 -BSA-FITC to this membrane-localized form of the ER would further establish that E 2  conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E 2 -BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E 2  to HeLa-ERα, but not native HeLa cells (figure  2 ). Scatchard analysis of the binding of E 2  to HeLa-ERα cells showed that although weakly expressed, the Kd for the expressed ERα was 7.04 nM, similar to published values (figure  3 ). HeLa-ERα cells, but not native HeLa cells, exhibited fluorescent staining of the cell membrane after incubation with E 2 -BSA-FITC (figure  4 ). The heterogeneous staining pattern reflected the low level of ERα expression. This fluorescence was not seen when HeLa-ERα cells were incubated with BSA conjugated to fluorescein alone (data not shown).To establish the specificity of E 2 -BSA binding, MC7 cells and HeLa-ERα cells were incubated with E 2 -BSA-FITC after pre-incubation with various concentrations of E 2  and the anti-estrogen ICI-182,780. As shown in figure  5 , fluorescence was lost in both cell types in a dose dependent manner with increasing concentrations of E 2 . Fluorescence was almost completely eliminated by pre-incubation with the specific competitor ICI-182,780. BSA conjugated to FITC alone did not bind. These results suggest that estrogen covalently bound to BSA can bind to ER in a biologically significant manner.The possibility that E 2 -BSA-FITC could be degraded during incubation with intact cells was examined using HPLC. E 2 -BSA-FITC was incubated in empty wells or wells containing MC7 cells under the same conditions employed for the binding studies described above. Media was removed from the cells and HPLC performed with a reverse phase column. Peaks were visualized using a scanning fluorescence detector. Aqueous solutions of E 2 -BSA-FITC produced a single peak with a retention time of 5.5 minutes using a methanol-water gradient from 80% to 50% over 30 minutes at 1 ml/minute. E 2  and E 2 -BSA did not fluoresce at the excitation and emission wavelengths used (data not shown). Spectra obtained from media containing E 2 -BSA-FITC alone and media containing E 2 -BSA-FITC incubated with MC7 cells are shown in figure  6 . The average area under the curve for E 2 -BSA-FITC was the same (p < 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E 2 -BSA-FITC is stable under the culture conditions employed for the binding experiments.Discussion The cellular effects elicited by estrogen [ 11 , 12 , 18 - 20 , 3 ] testosterone [ 3 , 21 , 22 ] and progesterone [ 23 - 25 ] covalently conjugated to membrane impermeable BSA have been attributed to non-genomic actions mediated by membrane associated hormone receptors. The use of these reagents for this purpose remains controversial for several reasons. A recent report demonstrated that E 2 -BSA does not bind to purified ER in competition assays with labeled E 2  [ 14 ]. The studies were performed when E 2 -BSA or cold E 2  were added concurrently with labeled E 2 . We obtained similar results under these conditions. However, pre-incubation of E 2 -BSA with purified ER results in significant competition with labeled E 2 . These conflicting results may be explained by differences in the rate of binding between E 2  and E 2 -BSA. E 2 -BSA is a large, bulky molecule similar in size to the ER and is probably spherical in general structure as is the parent BSA molecule. The BSA protein conformation immediately adjacent to the covalently bound estrogen undoubtedly provides substantial steric hindrance to the proper presentation of conjugated E 2  to the binding pocket of ER. The increased size of the E 2 -BSA molecule would also reduce the rate of its diffusion compared with the smaller E 2 . Correct orientation of E2 in the ER binding pocket is also impeded by the restraint on three-dimensional movement imposed by the six atom spacer used to connect BSA and E 2 . Lastly, the use of E 2 -BSA solutions that are formulated in terms of the molarity of total bound-E 2  probably overestimates the amount of E 2  available for binding. The rate of binding between E 2  and ER can be expressed using the second order rate equation: rate = k [E 2 ] [ER], where [E 2 ] is the concentration of estradiol, [ER] is the concentration of the ER, and k the rate constant. Commercially available E 2 -BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E 2 -BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E 2 -BSA with respect to the concentration of estradiol alone. However the rate of E 2 -BSA binding is dependent upon the concentration of E 2 -BSA (rate = k [E 2 -BSA] [ER]). Even if every collision between E 2 -BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate. Taken together, these factors reduce the binding efficiency of E 2 -BSA to ER compared with free E 2 . However, once binding has occurred, the stability of the E 2  molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E 2 -BSA results in successful binding, whereas immediate addition of E 2 -BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators [ 15 ] that demonstrate specific cell surface binding of E 2 -BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E 2 -BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E 2 -BSA and ER. Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than E 2 -BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells [ 26 ]. Membrane specific staining was prevented by treatment with antisense ERα mRNA or peptides that interfere with antibody binding. E 2  conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E 2  [ 27 ]. The membrane impermeable E 2 -BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ [ 15 ]. Moreover, ERα and ERβ interact directly with the membrane associated Src complex to trigger prostate cancer cell proliferation through the RAF-1/Erk-2 signal transduction pathway [ 5 ]. Lastly, we demonstrate that E 2 -BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E 2  are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered.
Discussion
The cellular effects elicited by estrogen [ 11 , 12 , 18 - 20 , 3 ] testosterone [ 3 , 21 , 22 ] and progesterone [ 23 - 25 ] covalently conjugated to membrane impermeable BSA have been attributed to non-genomic actions mediated by membrane associated hormone receptors. The use of these reagents for this purpose remains controversial for several reasons. A recent report demonstrated that E 2 -BSA does not bind to purified ER in competition assays with labeled E 2  [ 14 ]. The studies were performed when E 2 -BSA or cold E 2  were added concurrently with labeled E 2 . We obtained similar results under these conditions. However, pre-incubation of E 2 -BSA with purified ER results in significant competition with labeled E 2 . These conflicting results may be explained by differences in the rate of binding between E 2  and E 2 -BSA. E 2 -BSA is a large, bulky molecule similar in size to the ER and is probably spherical in general structure as is the parent BSA molecule. The BSA protein conformation immediately adjacent to the covalently bound estrogen undoubtedly provides substantial steric hindrance to the proper presentation of conjugated E 2  to the binding pocket of ER. The increased size of the E 2 -BSA molecule would also reduce the rate of its diffusion compared with the smaller E 2 . Correct orientation of E2 in the ER binding pocket is also impeded by the restraint on three-dimensional movement imposed by the six atom spacer used to connect BSA and E 2 . Lastly, the use of E 2 -BSA solutions that are formulated in terms of the molarity of total bound-E 2  probably overestimates the amount of E 2  available for binding. The rate of binding between E 2  and ER can be expressed using the second order rate equation: rate = k [E 2 ] [ER], where [E 2 ] is the concentration of estradiol, [ER] is the concentration of the ER, and k the rate constant.Commercially available E 2 -BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E 2 -BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E 2 -BSA with respect to the concentration of estradiol alone. However the rate of E 2 -BSA binding is dependent upon the concentration of E 2 -BSA (rate = k [E 2 -BSA] [ER]). Even if every collision between E 2 -BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate.Taken together, these factors reduce the binding efficiency of E 2 -BSA to ER compared with free E 2 . However, once binding has occurred, the stability of the E 2  molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E 2 -BSA results in successful binding, whereas immediate addition of E 2 -BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators [ 15 ] that demonstrate specific cell surface binding of E 2 -BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E 2 -BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E 2 -BSA and ER.Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than E 2 -BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells [ 26 ]. Membrane specific staining was prevented by treatment with antisense ERα mRNA or peptides that interfere with antibody binding. E 2  conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E 2  [ 27 ]. The membrane impermeable E 2 -BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ [ 15 ]. Moreover, ERα and ERβ interact directly with the membrane associated Src complex to trigger prostate cancer cell proliferation through the RAF-1/Erk-2 signal transduction pathway [ 5 ]. Lastly, we demonstrate that E 2 -BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E 2  are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered.Conclusions The results presented here suggest that E 2 -BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E 2  and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein.
Conclusions
The results presented here suggest that E 2 -BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E 2  and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein.Materials and Methods Establishment of ER stable transfectants Full-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml). Preparation of E 2 -BSA free of E 2 400 ul of E 2 -BSA (10 -5  M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul. Binding of estradiol to purified estrogen receptor 3 H-labeled E 2  (NEN, specific activity 48 Ci/mmol, 10 -8  M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E 2  to ERα was competed by various concentrations of ultrafiltered E 2 -BSA or E 2  (10 -9  to 3.5 × 10 -6  M in E 2 ) added four hours prior to or concurrently with the addition of labeled E 2 . ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and  3 H-E 2  binding determined by liquid scintillation counting. E 2 -BSA-FITC binding to cell membranes of ER producing cells HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 10 -8  M (in estrogen) E 2 -BSA conjugated to FITC (E 2 -BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E 2 -BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E 2 -BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E 2 , ICI-182,780, or E 2 -BSA (Sigma, 10 -7  to 10 -9  M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp). The stability of E 2 -BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E 2 -BSA-FITC (500 ul, 10 -8  M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate. Estradiol binding studies Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10 6  cells) incubated with several concentrations of  3 H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis.
Materials and Methods
Establishment of ER stable transfectants Full-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml).
Establishment of ER stable transfectants
Full-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml).Preparation of E 2 -BSA free of E 2 400 ul of E 2 -BSA (10 -5  M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul.
Preparation of E 2 -BSA free of E 2
400 ul of E 2 -BSA (10 -5  M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul.Binding of estradiol to purified estrogen receptor 3 H-labeled E 2  (NEN, specific activity 48 Ci/mmol, 10 -8  M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E 2  to ERα was competed by various concentrations of ultrafiltered E 2 -BSA or E 2  (10 -9  to 3.5 × 10 -6  M in E 2 ) added four hours prior to or concurrently with the addition of labeled E 2 . ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and  3 H-E 2  binding determined by liquid scintillation counting.
Binding of estradiol to purified estrogen receptor
3 H-labeled E 2  (NEN, specific activity 48 Ci/mmol, 10 -8  M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E 2  to ERα was competed by various concentrations of ultrafiltered E 2 -BSA or E 2  (10 -9  to 3.5 × 10 -6  M in E 2 ) added four hours prior to or concurrently with the addition of labeled E 2 . ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and  3 H-E 2  binding determined by liquid scintillation counting.E 2 -BSA-FITC binding to cell membranes of ER producing cells HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 10 -8  M (in estrogen) E 2 -BSA conjugated to FITC (E 2 -BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E 2 -BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E 2 -BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E 2 , ICI-182,780, or E 2 -BSA (Sigma, 10 -7  to 10 -9  M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp). The stability of E 2 -BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E 2 -BSA-FITC (500 ul, 10 -8  M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate.
E 2 -BSA-FITC binding to cell membranes of ER producing cells
HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 10 -8  M (in estrogen) E 2 -BSA conjugated to FITC (E 2 -BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E 2 -BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E 2 -BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E 2 , ICI-182,780, or E 2 -BSA (Sigma, 10 -7  to 10 -9  M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp).The stability of E 2 -BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E 2 -BSA-FITC (500 ul, 10 -8  M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate.Estradiol binding studies Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10 6  cells) incubated with several concentrations of  3 H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis.
Estradiol binding studies
Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10 6  cells) incubated with several concentrations of  3 H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis.Competing interests None declared.
Competing interests
None declared.Authors' contributions DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays.
Authors' contributions
DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays.


Background Smac/Diablo is a recently identified protein that is released from mitochondria after apoptotic stimuli. It binds IAPs, allowing caspase activation and cell death. In view of its activity it might participate in carcinogenesis. In the present study, we analyzed Smac expression in a panel of cervical cancer patients.
Background
Smac/Diablo is a recently identified protein that is released from mitochondria after apoptotic stimuli. It binds IAPs, allowing caspase activation and cell death. In view of its activity it might participate in carcinogenesis. In the present study, we analyzed Smac expression in a panel of cervical cancer patients.Methods We performed semi quantitative RT-PCR on 41 cervical tumor and 6 normal tissue samples. The study included 8 stage I cases; 16 stage II; 17 stage III; and a control group of 6 samples of normal cervical squamous epithelial tissue.
Methods
We performed semi quantitative RT-PCR on 41 cervical tumor and 6 normal tissue samples. The study included 8 stage I cases; 16 stage II; 17 stage III; and a control group of 6 samples of normal cervical squamous epithelial tissue.Results Smac mRNA expression was below the detection limit in the normal cervical tissue samples. In contrast, 13 (31.7%) of the 41 cervical cancer biopsies showed detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages (5 in stage I, 4 in stage II and 4 in stage III) with similar expression levels. We found no correlation between the presence of Smac mRNA and histology, menopause, WHO stage or disease status.
Results
Smac mRNA expression was below the detection limit in the normal cervical tissue samples. In contrast, 13 (31.7%) of the 41 cervical cancer biopsies showed detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages (5 in stage I, 4 in stage II and 4 in stage III) with similar expression levels. We found no correlation between the presence of Smac mRNA and histology, menopause, WHO stage or disease status.Conclusions Smac is expressed  de novo  in a subset of cervical cancer patients, reflecting a possible heterogeneity in the pathways leading to cervical cancer. There was no correlation with any clinical variable.
Conclusions
Smac is expressed  de novo  in a subset of cervical cancer patients, reflecting a possible heterogeneity in the pathways leading to cervical cancer. There was no correlation with any clinical variable.de novo

Background Apoptosis is an evolutionarily conserved biological process that plays a fundamental role in development and tissue homeostasis in metazoans [ 1 ]. This type of cell death is executed by a family of proteases known as caspases [ 2 ]. There are two well-characterized apoptotic pathways that converge in caspase activation: the death receptor pathway and the mitochondrial pathway [ 3 ]. Inhibitors of Apoptosis Proteins (IAPs) are the most important regulators of caspases. These proteins inhibit caspase activation, thus preventing the induction of apoptosis [ 4 ]. In cells undergoing apoptosis, IAPs are inactivated by interaction with proteins containing the so-called IBM (IAP-binding motif) [ 4 , 5 ]. One IBM protein is the recently identified Smac/DIABLO [ 6 , 7 ]. Smac resides in the mitochondrial intermembrane space in healthy cells but is released into the cytosol during apoptosis, where it interacts with IAPs and disrupts their ability to bind caspases [ 8 ]. Smac is expressed ubiquitously, with high expression in adult testis, heart, liver, kidney, spleen, prostate and ovary and low expression in brain, lung, thymus, and peripheral blood leukocytes [ 9 ]. It is encoded in a nuclear gene and is post-translationally imported into the mitochondria via a targeting sequence in its amino terminus. Removal of this signal generates a mature polypeptide with the IBM at the amino terminal end [ 10 ]. Smac interacts with all mammalian IAPs examined so far: XIAP, cIAP-1, cIAP-2, survivin and ML-IAP [ 6 , 7 , 11 , 12 ]. The structure of the Smac-XIAP complex has been studied by X-ray crystallography [ 13 ] and high-resolution NMR [ 14 ]; it appears that the tetrapeptide AVPI is indispensable for the formation of this complex. IAPs are highly expressed in human tumor cells [ 15 - 17 ], contributing to the intrinsic resistance of these cells to endogenous death receptor-induced apoptosis and consequently to chemotherapy [ 18 ]. For this reason, peptides mimicking the action of Smac have been generated and analyzed. Four publications to date have shown promising effects of these Smac peptides  in vitro  and  in vivo ; however, further studies are required prior to clinical testing [ 19 - 22 ]. Recently, Sekimura and colleagues found that Smac expression was significantly lower in primary lung cancers than in normal tissue [ 23 ]; patients with lower Smac mRNA levels had worse prognoses. These results indicate that Smac expression may play a role in the progression of primary lung cancer and may be useful for prognosis [ 23 ]. However, Smac expression has not been analyzed in other tumors. In view of the possible role of Smac in cervical carcinogenesis and its potential as a therapeutic target, we have investigated the expression of this apoptotic protein in cervical cancer patients.
Background
Apoptosis is an evolutionarily conserved biological process that plays a fundamental role in development and tissue homeostasis in metazoans [ 1 ]. This type of cell death is executed by a family of proteases known as caspases [ 2 ]. There are two well-characterized apoptotic pathways that converge in caspase activation: the death receptor pathway and the mitochondrial pathway [ 3 ]. Inhibitors of Apoptosis Proteins (IAPs) are the most important regulators of caspases. These proteins inhibit caspase activation, thus preventing the induction of apoptosis [ 4 ]. In cells undergoing apoptosis, IAPs are inactivated by interaction with proteins containing the so-called IBM (IAP-binding motif) [ 4 , 5 ].One IBM protein is the recently identified Smac/DIABLO [ 6 , 7 ]. Smac resides in the mitochondrial intermembrane space in healthy cells but is released into the cytosol during apoptosis, where it interacts with IAPs and disrupts their ability to bind caspases [ 8 ]. Smac is expressed ubiquitously, with high expression in adult testis, heart, liver, kidney, spleen, prostate and ovary and low expression in brain, lung, thymus, and peripheral blood leukocytes [ 9 ]. It is encoded in a nuclear gene and is post-translationally imported into the mitochondria via a targeting sequence in its amino terminus. Removal of this signal generates a mature polypeptide with the IBM at the amino terminal end [ 10 ]. Smac interacts with all mammalian IAPs examined so far: XIAP, cIAP-1, cIAP-2, survivin and ML-IAP [ 6 , 7 , 11 , 12 ]. The structure of the Smac-XIAP complex has been studied by X-ray crystallography [ 13 ] and high-resolution NMR [ 14 ]; it appears that the tetrapeptide AVPI is indispensable for the formation of this complex.IAPs are highly expressed in human tumor cells [ 15 - 17 ], contributing to the intrinsic resistance of these cells to endogenous death receptor-induced apoptosis and consequently to chemotherapy [ 18 ]. For this reason, peptides mimicking the action of Smac have been generated and analyzed. Four publications to date have shown promising effects of these Smac peptides  in vitro  and  in vivo ; however, further studies are required prior to clinical testing [ 19 - 22 ].in vitroin vivoRecently, Sekimura and colleagues found that Smac expression was significantly lower in primary lung cancers than in normal tissue [ 23 ]; patients with lower Smac mRNA levels had worse prognoses. These results indicate that Smac expression may play a role in the progression of primary lung cancer and may be useful for prognosis [ 23 ]. However, Smac expression has not been analyzed in other tumors. In view of the possible role of Smac in cervical carcinogenesis and its potential as a therapeutic target, we have investigated the expression of this apoptotic protein in cervical cancer patients.Methods Cell lines and tumor samples Cervical cancer cell lines (HeLa, SiHa, CaSki and CaLo) were obtained from ATCC and cultured as monolayers in Dulbecco Modified Eagle's Medium (DMEM) containing 10% (V/V) fetal bovine serum (GIBCO, Bethesda, MD, USA) at 37°C in a humidified atmosphere of 5% (V/V) CO 2 . Forty-one cervical cancer samples were obtained from the Instituto Nacional de Cancerologia of Mexico. Written consent was obtained from patients before the samples were collected. Tumors were staged according to the International Gynecology and Obstetric Federation (FIGO) system. The samples comprised 8 at stage IB, 16 at stage IIB and 17 at stage IIIB; and a control group comprising 6 samples of normal cervical squamous epithelial tissue (Table  1 ). The control samples were derived from hysterectomy specimens from patients with uterine myomatosis. Only samples with normal pathological reports were included. Histology Histopathological grading was done according to the WHO (World Health Organization) classification system (Table  1 ). RNA isolation and RT-PCR RNA extraction and RT-PCR analysis were performed as described previously [ 24 ]. Briefly, total RNA was extracted from cultured cells, tumors and non-neoplastic tissue samples with Trizol reagent (Invitrogen) following the manufacturer's protocol. RNA purity was confirmed by the 260/280 nm absorbance ratio and its integrity was established with agarose gels. Total RNA (2 μg) was reverse-transcribed in a final 20 μl reaction volume using 15 U ThermoScript reverse transcriptase, 2.5 × RT Buffer and random hexamers (ThermoScript RT-PCR, Invitrogen). The RT-PCR steps were 25°C for 10 min, 50°C for 50 min and 85°C for 5 min. Smac and GAPDH mRNA PCR reactions contained 0.25 μl Amplitaq gold polymerase (Applied Biosystems, ROCHE), 2.5 μl 10 × reaction buffer, 0.5 μl dNTP mix 10 mM, 1 μl sense primer 10 μM, 1 μl anti-sense primer 10 μM and 1 μl cDNA in 25 μl final volume. The Smac primers were: sense 5' GCGCGGATCCATGGCGGCTCTGAAGAGTTG 3' and anti-sense 5' AGCTCTCTAGACTCAGGCCCTCAATCCTCA 3'. The GAPDH primers were: sense 5' CCCCTTCATTGACCTCAACT 3' and antisense 5' TTGTCATGGATGACCTTGGC 3'. The PCR cycle parameters for Smac were: 10 min enzyme activation at 95°C followed by 3 cycles of 30 s at 95°C and 2 min at 72°C, then 30 cycles of 30 s at 95°C and 30 s at 68°C, and finally 5 min at 72°C. The corresponding parameters for GAPDH were: 10 min enzyme activation at 95°C followed by 25 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. The products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Smac mRNA data were expressed as ratios between the densitometric values (Scion Image software) of Smac gene expression. The PCR products were normalized to the amplified GAPDH, the internal reference gene. Gene expression measurements were repeated at least twice. Statistical analysis To detect a correlation between pathological tumor parameters and normalized Smac expression we used ANOVA (stage, current disease and menopause status) and  chi  square tests (stage, histology of tumors, menopause and current status). Kaplan-Meier curves for status were generated and log rank was used to test for differences. The mean follow-up was 14.7 months. The statistical package Intercooled Stata 7.0 was used for analyses and statistical significance was accepted when the  p  value was less than 0.05.
Methods
Cell lines and tumor samples Cervical cancer cell lines (HeLa, SiHa, CaSki and CaLo) were obtained from ATCC and cultured as monolayers in Dulbecco Modified Eagle's Medium (DMEM) containing 10% (V/V) fetal bovine serum (GIBCO, Bethesda, MD, USA) at 37°C in a humidified atmosphere of 5% (V/V) CO 2 . Forty-one cervical cancer samples were obtained from the Instituto Nacional de Cancerologia of Mexico. Written consent was obtained from patients before the samples were collected. Tumors were staged according to the International Gynecology and Obstetric Federation (FIGO) system. The samples comprised 8 at stage IB, 16 at stage IIB and 17 at stage IIIB; and a control group comprising 6 samples of normal cervical squamous epithelial tissue (Table  1 ). The control samples were derived from hysterectomy specimens from patients with uterine myomatosis. Only samples with normal pathological reports were included.
Cell lines and tumor samples
Cervical cancer cell lines (HeLa, SiHa, CaSki and CaLo) were obtained from ATCC and cultured as monolayers in Dulbecco Modified Eagle's Medium (DMEM) containing 10% (V/V) fetal bovine serum (GIBCO, Bethesda, MD, USA) at 37°C in a humidified atmosphere of 5% (V/V) CO 2 .Forty-one cervical cancer samples were obtained from the Instituto Nacional de Cancerologia of Mexico. Written consent was obtained from patients before the samples were collected. Tumors were staged according to the International Gynecology and Obstetric Federation (FIGO) system. The samples comprised 8 at stage IB, 16 at stage IIB and 17 at stage IIIB; and a control group comprising 6 samples of normal cervical squamous epithelial tissue (Table  1 ). The control samples were derived from hysterectomy specimens from patients with uterine myomatosis. Only samples with normal pathological reports were included.Histology Histopathological grading was done according to the WHO (World Health Organization) classification system (Table  1 ).
Histology
Histopathological grading was done according to the WHO (World Health Organization) classification system (Table  1 ).RNA isolation and RT-PCR RNA extraction and RT-PCR analysis were performed as described previously [ 24 ]. Briefly, total RNA was extracted from cultured cells, tumors and non-neoplastic tissue samples with Trizol reagent (Invitrogen) following the manufacturer's protocol. RNA purity was confirmed by the 260/280 nm absorbance ratio and its integrity was established with agarose gels. Total RNA (2 μg) was reverse-transcribed in a final 20 μl reaction volume using 15 U ThermoScript reverse transcriptase, 2.5 × RT Buffer and random hexamers (ThermoScript RT-PCR, Invitrogen). The RT-PCR steps were 25°C for 10 min, 50°C for 50 min and 85°C for 5 min. Smac and GAPDH mRNA PCR reactions contained 0.25 μl Amplitaq gold polymerase (Applied Biosystems, ROCHE), 2.5 μl 10 × reaction buffer, 0.5 μl dNTP mix 10 mM, 1 μl sense primer 10 μM, 1 μl anti-sense primer 10 μM and 1 μl cDNA in 25 μl final volume. The Smac primers were: sense 5' GCGCGGATCCATGGCGGCTCTGAAGAGTTG 3' and anti-sense 5' AGCTCTCTAGACTCAGGCCCTCAATCCTCA 3'. The GAPDH primers were: sense 5' CCCCTTCATTGACCTCAACT 3' and antisense 5' TTGTCATGGATGACCTTGGC 3'. The PCR cycle parameters for Smac were: 10 min enzyme activation at 95°C followed by 3 cycles of 30 s at 95°C and 2 min at 72°C, then 30 cycles of 30 s at 95°C and 30 s at 68°C, and finally 5 min at 72°C. The corresponding parameters for GAPDH were: 10 min enzyme activation at 95°C followed by 25 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. The products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Smac mRNA data were expressed as ratios between the densitometric values (Scion Image software) of Smac gene expression. The PCR products were normalized to the amplified GAPDH, the internal reference gene. Gene expression measurements were repeated at least twice.
RNA isolation and RT-PCR
RNA extraction and RT-PCR analysis were performed as described previously [ 24 ]. Briefly, total RNA was extracted from cultured cells, tumors and non-neoplastic tissue samples with Trizol reagent (Invitrogen) following the manufacturer's protocol. RNA purity was confirmed by the 260/280 nm absorbance ratio and its integrity was established with agarose gels. Total RNA (2 μg) was reverse-transcribed in a final 20 μl reaction volume using 15 U ThermoScript reverse transcriptase, 2.5 × RT Buffer and random hexamers (ThermoScript RT-PCR, Invitrogen). The RT-PCR steps were 25°C for 10 min, 50°C for 50 min and 85°C for 5 min. Smac and GAPDH mRNA PCR reactions contained 0.25 μl Amplitaq gold polymerase (Applied Biosystems, ROCHE), 2.5 μl 10 × reaction buffer, 0.5 μl dNTP mix 10 mM, 1 μl sense primer 10 μM, 1 μl anti-sense primer 10 μM and 1 μl cDNA in 25 μl final volume. The Smac primers were: sense 5' GCGCGGATCCATGGCGGCTCTGAAGAGTTG 3' and anti-sense 5' AGCTCTCTAGACTCAGGCCCTCAATCCTCA 3'. The GAPDH primers were: sense 5' CCCCTTCATTGACCTCAACT 3' and antisense 5' TTGTCATGGATGACCTTGGC 3'.The PCR cycle parameters for Smac were: 10 min enzyme activation at 95°C followed by 3 cycles of 30 s at 95°C and 2 min at 72°C, then 30 cycles of 30 s at 95°C and 30 s at 68°C, and finally 5 min at 72°C. The corresponding parameters for GAPDH were: 10 min enzyme activation at 95°C followed by 25 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. The products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Smac mRNA data were expressed as ratios between the densitometric values (Scion Image software) of Smac gene expression. The PCR products were normalized to the amplified GAPDH, the internal reference gene. Gene expression measurements were repeated at least twice.Statistical analysis To detect a correlation between pathological tumor parameters and normalized Smac expression we used ANOVA (stage, current disease and menopause status) and  chi  square tests (stage, histology of tumors, menopause and current status). Kaplan-Meier curves for status were generated and log rank was used to test for differences. The mean follow-up was 14.7 months. The statistical package Intercooled Stata 7.0 was used for analyses and statistical significance was accepted when the  p  value was less than 0.05.
Statistical analysis
To detect a correlation between pathological tumor parameters and normalized Smac expression we used ANOVA (stage, current disease and menopause status) and  chi  square tests (stage, histology of tumors, menopause and current status). Kaplan-Meier curves for status were generated and log rank was used to test for differences. The mean follow-up was 14.7 months. The statistical package Intercooled Stata 7.0 was used for analyses and statistical significance was accepted when the  p  value was less than 0.05.chipResults To ascertain whether Smac is expressed in cervical cancer we performed semiquantitative RT-PCR analyses on a panel of cervical cancer lines, including HeLa, SiHa, CasKi and CaLo cells. As shown in Figure  1 , the HeLa and CasKi lines contained Smac mRNA, but very low levels were observed in SiHa and CaLo cells. Next, we measured Smac mRNA levels using the same approach in 41 cervical tumor and 6 normal cervical samples. To ensure accurate determinations and to verify equal RNA input, GAPDH mRNA was amplified simultaneously. Figure  2  shows a representative panel of results, which are given in Tables  1  and  2 . Unexpectedly, Smac mRNA was below the detection limit in normal cervical samples. In contrast, as expected from the cell line data, 13 (31.7%) of the 41 cervical cancer biopsies contained detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages (5 in stage I, 4 in stage II and 4 in stage III). We found no significant correlation between Smac mRNA level and histology, menopause, clinical stage or disease status (Table  2 ). When the Smac expression levels in the tumor samples were analyzed, there were no significant differences between clinical stages (Figure  3 ), menopause status (Figure  4 ) or disease status (Figure  5 ). Similarly, a survival analysis of the patients showed no statistical differences between patients expressing or not expressing Smac mRNA (Figure  6 ).
Results
To ascertain whether Smac is expressed in cervical cancer we performed semiquantitative RT-PCR analyses on a panel of cervical cancer lines, including HeLa, SiHa, CasKi and CaLo cells. As shown in Figure  1 , the HeLa and CasKi lines contained Smac mRNA, but very low levels were observed in SiHa and CaLo cells.Next, we measured Smac mRNA levels using the same approach in 41 cervical tumor and 6 normal cervical samples. To ensure accurate determinations and to verify equal RNA input, GAPDH mRNA was amplified simultaneously. Figure  2  shows a representative panel of results, which are given in Tables  1  and  2 . Unexpectedly, Smac mRNA was below the detection limit in normal cervical samples. In contrast, as expected from the cell line data, 13 (31.7%) of the 41 cervical cancer biopsies contained detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages (5 in stage I, 4 in stage II and 4 in stage III). We found no significant correlation between Smac mRNA level and histology, menopause, clinical stage or disease status (Table  2 ). When the Smac expression levels in the tumor samples were analyzed, there were no significant differences between clinical stages (Figure  3 ), menopause status (Figure  4 ) or disease status (Figure  5 ). Similarly, a survival analysis of the patients showed no statistical differences between patients expressing or not expressing Smac mRNA (Figure  6 ).Discussion Tumors proliferate beyond the constraints that limit growth in normal tissue. Therefore, the resistance of tumor cells to apoptosis is an essential feature of carcinogenesis. This has been confirmed by the finding that deregulated proliferation alone is not sufficient for tumor formation because there is concomitant induction of cell death [ 25 ]. Overexpression of growth-promoting oncogenes such as c-Myc sensitize cells to apoptosis [ 26 ]. Thus, tumor progression requires the expression of anti-apoptotic proteins or the inactivation of essential pro apoptotic proteins [ 27 , 28 ]. Indeed, it has been shown that survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family, is upregulated in some tumors [ 29 ], correlating with prognosis [ 30 , 31 ]. Smac is a recently identified proapoptotic protein that interacts with and inhibits several IAPs, including survivin [ 6 , 11 ]. It has been shown that Smac mRNA levels in tumor tissues are significantly lower than in normal tissues [ 23 ]. Patients with lower Smac mRNA levels have worse prognoses. These results indicate that Smac expression may play a role in the progression of primary lung cancer, as expected by the known role of this protein in cell death induced by chemotherapeutic drugs. Unexpectedly, we found that during cancer progression, some cervical tumors express this protein  de novo . Unfortunately, we found no correlation between Smac expression and any clinical variable. This could be attributed to differences in tissue expression of IAPs, which are reported to have different binding affinities for Smac. On the other hand, alternative IAPs such as the recently identified Omi/Htra2 [ 32 ] might play an important tissue- or tumor-specific role. This is supported by the recent report of a null phenotype in Smac-deficient mice, in which a role for other IAP inhibitory proteins is suspected [ 33 ]. Cancer treatment by chemotherapy and γ-irradiation kills cells primarily by the induction of apoptosis. However, few tumors are wholly sensitive to these therapies, and the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells [ 34 ]. Modulation of the key elements in apoptotic signaling should directly influence therapy-induced tumor-cell death. Indeed, it has recently been suggested that peptides mimicking the Smac amino-terminus could be a novel therapeutic weapon [ 19 ]. Tumors with low or null Smac expression, such as the ones reported in this study, could be more susceptible to this approach.
Discussion
Tumors proliferate beyond the constraints that limit growth in normal tissue. Therefore, the resistance of tumor cells to apoptosis is an essential feature of carcinogenesis. This has been confirmed by the finding that deregulated proliferation alone is not sufficient for tumor formation because there is concomitant induction of cell death [ 25 ]. Overexpression of growth-promoting oncogenes such as c-Myc sensitize cells to apoptosis [ 26 ]. Thus, tumor progression requires the expression of anti-apoptotic proteins or the inactivation of essential pro apoptotic proteins [ 27 , 28 ]. Indeed, it has been shown that survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family, is upregulated in some tumors [ 29 ], correlating with prognosis [ 30 , 31 ].Smac is a recently identified proapoptotic protein that interacts with and inhibits several IAPs, including survivin [ 6 , 11 ]. It has been shown that Smac mRNA levels in tumor tissues are significantly lower than in normal tissues [ 23 ]. Patients with lower Smac mRNA levels have worse prognoses. These results indicate that Smac expression may play a role in the progression of primary lung cancer, as expected by the known role of this protein in cell death induced by chemotherapeutic drugs. Unexpectedly, we found that during cancer progression, some cervical tumors express this protein  de novo .de novoUnfortunately, we found no correlation between Smac expression and any clinical variable. This could be attributed to differences in tissue expression of IAPs, which are reported to have different binding affinities for Smac. On the other hand, alternative IAPs such as the recently identified Omi/Htra2 [ 32 ] might play an important tissue- or tumor-specific role. This is supported by the recent report of a null phenotype in Smac-deficient mice, in which a role for other IAP inhibitory proteins is suspected [ 33 ].Cancer treatment by chemotherapy and γ-irradiation kills cells primarily by the induction of apoptosis. However, few tumors are wholly sensitive to these therapies, and the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells [ 34 ]. Modulation of the key elements in apoptotic signaling should directly influence therapy-induced tumor-cell death. Indeed, it has recently been suggested that peptides mimicking the Smac amino-terminus could be a novel therapeutic weapon [ 19 ]. Tumors with low or null Smac expression, such as the ones reported in this study, could be more susceptible to this approach.Conclusions During cervical cancer progression, a subset of tumors express the apoptotic protein Smac  de novo . This finding contrasts with a previous report for lung cancer [ 23 ], underlining the notion that downregulation or even expression of Smac could be dispensable for tumor progression, at least in cervical cancer. This could be because other mitochondrial molecules such as Omi might substitute for its known proapoptotic function. There was no correlation between Smac expression and any clinical variable.
Conclusions
During cervical cancer progression, a subset of tumors express the apoptotic protein Smac  de novo . This finding contrasts with a previous report for lung cancer [ 23 ], underlining the notion that downregulation or even expression of Smac could be dispensable for tumor progression, at least in cervical cancer. This could be because other mitochondrial molecules such as Omi might substitute for its known proapoptotic function. There was no correlation between Smac expression and any clinical variable.de novoCompeting interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions JMZ Conceived and coordinated the study. VAML: Conceived and coordinated the study. Statistical Analysis MEC: Performed RT-PCR assays DCL: Provided the clinical samples and coordinated patient study CMLG: Coordinated patient assessment, ethical guidelines. JGGS: Provided clinical assessment
Authors' contributions
JMZ Conceived and coordinated the study.VAML: Conceived and coordinated the study. Statistical AnalysisMEC: Performed RT-PCR assaysDCL: Provided the clinical samples and coordinated patient studyCMLG: Coordinated patient assessment, ethical guidelines.JGGS: Provided clinical assessmentPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: