Mol. Hum. Reprod. Advance Access originally published online on July 26, 2006
Molecular Human Reproduction 2006 12(9):535-541; doi:10.1093/molehr/gah260
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Caspase cascade of Fas-mediated apoptosis in human normal endometrium and endometrial carcinoma cells
Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Osaka, Japan
1 To whom correspondence should be addressed at: Department of Anatomy and Cell Biology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. E-mail: an1001{at}art.osaka-med.ac.jp
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Abstract |
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Human endometrial epithelial cells undergo apoptosis immediately before the menstrual period. Apoptotic signalling was analysed using human endometrial tissue and a human endometrial carcinoma cell line (HHUA). Activity levels of caspase-3, -8, and -9 were elevated in human endometrium during the late secretory phase and in HHUA cells incubated with an anti-Fas monoclonal antibody (mAb). Fas-mediated apoptosis of HHUA cells was blocked by prior exposure to inhibitors of caspase-9, -8 and -3. In HHUA cells treated with anti-Fas mAb, a release of cytochrome c was detected in the cytosolic fraction, in addition a full-length Bid was degraded. Full-length FLIPL (p55) was degraded during apoptosis, and p29 (regarded as the product of p55 cleavage) appeared instead of FLIPL. In normal human endometrial tissue, Bid degradation was also observed in a cyclic manner with a peak during the early secretory phase of the menstrual cycle. Furthermore, the release of cytochrome c was seen in the early secretory phase. However, expression of FLIPS was only observed during the menstrual cycle in normal endometrial tissue. We concluded that the main apoptotic signalling in both normal human endometrial tissue and HHUA cells exposed to anti-Fas mAb is the mitochondrial pathway via Bid degradation. Although the function of FLIP is still unknown on normal endometrial tissue, it may be regulated by FLIPL expression on HHUA cells derived from human endometrial carcinoma.
Key words: apoptosis/Bid/caspase family/human endometrial carcinoma cell line/human endometrium
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Introduction |
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Menstruation was originally regarded as the ischaemic necrosis of a functional layer of the endometrium caused by the contraction of spiral arteries and dependent on the sex hormone concentration (Markee, 1940
; Bartelmez, 1957). However, electron microscopic studies later revealed apoptotic cells in human endometrial epithelial cells during the late secretory phase (Hopwood and Levison, 1976). Bcl-2 is an inhibitory factor of apoptosis (Tsujimoto, 1989; Garcia et al., 1992) that is expressed in human endometrial glandular cells during the proliferative phase but not during the late secretory phase (Otsuki et al., 1994). In contrast to the high level of Bcl-2 expression in human endometrial glandular cells during the proliferative phase, both Fas and Fas ligand (FasL) are expressed throughout the menstrual cycle (Yamashita et al., 1999). Immunoelectron microscopy of both Fas and FasL (Yamashita et al., 1999) showed that their subcellular localization in human endometrial glandular cells changed during the menstrual cycle. Both Fas and FasL were mainly localized on the Golgi apparatus during the late proliferative phase as well as on apical membranes and Golgi-transporting vesicles during the late secretory phase. Therefore, these studies suggested that apoptosis of human endometrial glandular cells is blocked by Bcl-2 expression during the proliferative phase and induced by the Fas/FasL system in an autocrine or paracrine manner during the secretory phase. However, it is generally accepted that Bcl-2 blocks apoptosis via the mitochondrial pathway and not the death receptor pathway induced by the Fas/FasL system. Details of the downstream signalling of the Fas/FasL system in human endometrial glandular cells during the menstrual cycle remain unknown.
Caspase-8 is activated by the Fas/FasL signal, resulting in the caspase cascade (Medema et al., 1997; Muzio et al., 1997). Two pathways are regulated by caspase-8 (Scaffidi et al., 1998). In one, procaspase-3 is directly cleaved by the active form of caspase-8 and transmits apoptotic signals by degrading target molecules. In the other, which is via the mitochondrial pathway, caspase-8 degrades Bid, which is a member of the Bcl-2 family (Wang et al., 1996), and the active Bid fragment localized in the mitochondria induces the release of cytochrome c from the mitochondrial membrane into the cytosol (Li et al., 1998; Luo et al., 1998). Cytosolic cytochrome c interacts with Apaf-1 and procaspase-9 to form an apoptosome (Li et al., 1997; Zou et al., 1999), and caspase-9 activated by autoprocessing cleaves procaspase-3 into its active form.
The purpose of this study was to clarify whether these two pathways are involved in human endometrial apoptosis, using human endometrium and a human endometrial carcinoma cell line (HHUA cells) (Ishiwata et al., 1984b). HHUA cells are sensitive to anti-Fas monoclonal antibody (mAb) and undergo apoptosis in response to the Fas/FasL signal (Tanaka et al., 1995).
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Materials and methods |
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Specimens and cell line
Endometrial samples were obtained from 32 well-informed and consenting pre-menopausal non-pregnant women (age range, 29–53 years) who underwent hysterectomy for benign diseases. All women had regular menstrual cycles and were not on contraceptives before surgery. The endometrium was obtained from corpora of the uteri and classified by histological dating making reference to the women’s menstrual history, as early proliferative (n = 4), late proliferative (n = 9), early secretory (n = 11) and late secretory (n = 8) phases.
HHUA cells (the Riken Cell Bank, Tsukuba, Japan) were maintained in Ham’s F-12 (Sigma, St Louis, MO, USA) supplemented with 15% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin (all from GIBCO-BRL, Grand island, NY, USA). 17ß-estradiol (E2), progesterone (P4) or their combination (Sigma) was added to the culture medium to analyse whether female sex hormones had an affect on HHUA cells. For the combination treatment, P4 concentrations varied with 10 nM E2. Apoptosis was induced by adding a final concentration of 1–100 ng/ml of mouse anti-human Fas IgM mAb (anti-Fas mAb, MBL, Nagoya, Japan) to the culture medium. Cells were harvested 0–24 h thereafter.
Cell proliferation assay and caspase inhibitor study
HHUA cell proliferation was determined using CellTiter 96® AQueous One Solution Proliferation Assay (Promega, Madison, WI, USA). HHUA cells at the log phase were harvested using 0.25% trypsin/1 mM EDTA (GIBCO-BRL) and suspended in the culture medium. The cell suspension was dispensed into 96-well culture plates (3000 cells/well) and incubated overnight. The medium was changed to fresh culture medium (100 µl/well), then anti-Fas mAb was added to wells and the plates were further incubated overnight. The CellTiter 96® AQueous One Solution reagent was added to the wells (20 µl/well), and the plates were further incubated for 3 h. The reaction was stopped with 10% sodium dodecyl sulphate (SDS) 25 µl/well. The absorbance of the reactant in the wells at 490 nm was determined by a 96-well plate reader. The blanks consisted of wells without cells. We calculated cell viability as the ratio of the number of live anti-Fas mAb treated cells to that in untreated cells (%). We added the caspase inhibitors, Z-VAD-FMK for pan-caspases (Enzyme Systems Products, Livermore, CA, USA), Ac-IETD-CHO for caspase-8, Ac-LEHD-CHO for caspase-9 and Ac-DEVD-CHO for caspase-3 (all from Peptide Institute Inc., Osaka, Japan) to the wells 2 h before anti-Fas mAb, to determine inhibition.
RT–PCR
Total RNA of HHUA cells (treated and untreated with E2) were extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. The RNA was reacted with RQ1 RNase-Free DNase (Promega) to avoid contamination of genomic DNA. cDNA was prepared with SuperScriptTM II reverse transcriptase (Invitrogen) using oligo(dT) primer (Invitrogen). The reaction mixture was incubated at 42°C for 50 min. An aliquot of cDNA (from 100 ng RNA) was used as a template for PCR. PCR primers were specifically designed for the human estrogen receptor (ER) 
, ß, and progesterone receptor (PR) (common region of PRA and B, refer to Table I). As an internal control, a primer of ß-actin (Invitrogen) was used. PCR was carried out with the GeneAmp® PCR System 2700 (Applied Biosystems, Foster City, CA, USA) using Platinum® Taq DNA Polymerase (Invitrogen). Amplification was performed for 40 cycles with denaturation at 94°C (30 s), annealing at 55°C (30 s) and extension at 72°C (1 min). As a control, normal human endometrial tissues were used. Negative control experiments lacked cDNA substrate to check for the presence of exogenous contaminant DNA. RT–PCR products were resolved by electrophoresis on 2.0% agarose gels. Bands stained with ethidium bromide were visualized under UV light at 302 nm.
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Immunohistochemistry
HHUA cells (treated and untreated with E2) were cultured in the Lab-TekTM II Chamber SlideTM System (NUNC, Roskilde, Denmark) and fixed with 4% paraformaldehyde. Formalin-fixed paraffin-embedded sections of human endometrial tissues were incubated at 121°C for 15 min in citrate buffer (pH 6.0) to activate epitope. All sections were permealized with phosphate-buffered saline (PBS)-T (PBS containing 0.05% Tween-20). Rabbit anti-human ER (MC-20) antibody (Santa Cruz, Santa Cruz, CA, USA) was then used. The secondary antibodies were peroxidase-conjugated, and the immunoreaction was visualized by staining with the chromogen, 3',3'-diamino-benzidine tetrahydroxychloride (DAB). Hematoxylin was used as a nuclear counter stain.
DNA fragmentation
DNA in HHUA cells was analysed by DNA agarose gel electrophoresis. HHUA cells were suspended in lysis buffer [50 mM Tris–HCl (pH 7.8), 10 mM EDTA and 0.5% sodium N-lauroylsarcosinate]. The cell lysates were digested with 0.5 mg/ml RNase A (Sigma) at 50°C for 20 min followed by 0.5 mg/ml proteinase K (Sigma) at 50°C for 30 min and then resolved by electrophoresis on 2.0% agarose gels. Bands stained with ethidium bromide were visualized under UV light at 302 nm.
Caspase activity
Human endometrial tissue and HHUA cells were homogenized in lysis buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl and 1% Triton X-100], then the cells were sedimented by centrifugation and the supernatants were extracted. Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA), and then the extracts were mixed with reaction buffer. The reaction mixture contained 100 mM HEPES–KOH (pH 7.3), 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol (DTT), 2% dimethyl sulfoxide (DMSO), 0.2 mM synthetic peptide substrates for caspases (Ac-DEVD-pNA for caspase-3, Ac-IETD-pNA for caspase-8 and Ac-LEHD-pNA for caspase-9; Alexis Corporation, San Diego, CA, USA) and 200 µg of protein from the extracts in a total volume of 100 µl. Reactions proceeded at 37°C for 1 h. The amount of free pNa (yellow colour) cleaved by caspases and released from the substrate was measured by monitoring the absorbance at 405 nm. Caspase activities were expressed as optical density at 405 nm.
Western blots
Rabbit anti-human Bid antibody (Genzyme-Techne, Cambridge, MA, USA), mouse anti-human FLIP antibody (Santa Cruz) and mouse anti-cytochrome c antibody (clone 7H8.2C12, BD PharMingen, San Jose, CA, USA) were used for Western blotting. The secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody (all from Dako, Glostrup, Denmark). Goat anti-actin antibody (Santa Cruz) was used as a control and the secondary against this antibody was peroxidase-conjugated donkey anti-goat IgG antibody (Santa Cruz).
Samples were homogenized in lysis buffer [30 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 µg/ml pepstatin A, 0.2 mM TPCK, 0.1 mM TLCK and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was separated by centrifugation, and then the supernatant (extract) was collected. Protein concentrations in the extract were determined as described above. Total protein (50 µg) from the extract was separated by SDS-polyacrylamide gel electrophoresis (15% polyacrylamide gel) and then electroblotted onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was incubated for 1 h at room temperature in TBS-T [25 mM Tris–HCl (pH 7.4), 150 mM NaCl and 0.05% Tween-20] containing 2% non-fat dry milk, then overnight at 4°C in TBS-T containing 1 : 1000 diluted anti-human Bid antibody or 1 : 100 diluted anti-human FLIP antibody. The membrane was further incubated for 1 h at room temperature in TBS-T containing peroxidase-conjugated anti-rabbit IgG (diluted 1 : 1000 for Bid) or anti mouse IgG (diluted 1 : 2000 for FLIP). Immunoblots were visualized by staining with the chromogen, DAB. For detection of cytochrome c release from mitochondria to cytosol, samples were homogenized in lysis buffer [20 mM HEPES–KOH (pH 7.3), 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis-(ß-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM DTT, 0.1 mM PMSF, 5 µg/ml pepstatin A and 10 µg/ml leupeptin]. The nuclei were sedimented by centrifugation at 600g for 10 min at 4°C. The mitochondria and lysosomes were removed from the supernatant by centrifugation at 8000g for 10 min at 4°C. Finally, the supernatant was further centrifuged at 100 000g for 1 h at 4°C. The resulting supernatant (cytosolic fraction) was Western blotted as described above using a primary mouse anti-cytochrome c mAb diluted 1 : 500. The band densities were measured on membranes with the Scion Image (Scion Corporation, Frederick, MD, USA). Actin density served as the reference correction factor.
Statistical analysis
Quantitative data were analysed using a two-sided Student’s t-test. Significant values were evaluated at *P < 0.05 and **P < 0.01.
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Effects of female sex hormone and Fas-mediated cell death on HHUA cells
Proliferation of HHUA cells was significantly activated by estrogen (17ß-estradiol; E2) at a low concentration (100 pM) (Figure 1a). On the other hand, progesterone (P4) at over 100 nM slightly inhibited the cell proliferation at over 100 nM, and cell death did not occur. To examine the presence of female sex hormone receptors in HHUA cells, the expression of these hormone receptors was analysed. In human endometrial tissues, ER
and PR were expressed, but ERß was not (Figure 1b, left panel). On the other hand, HHUA cells expressed only ERß and had no change in the expression levels for E2 concentrations (Figure 1b, right panel). ER was also not detected by immunohistochemistry (Figure 1c). Treatment with progesterone (P4) and a combination of P4 and 10 nM E2 showed the same results as treatment with E2 alone (data not shown).
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Treatment with anti-Fas mAb decreased cell viability of HHUA cells (Figure 2a) and also dose-dependently induced DNA fragmentation (Figure 2b). Therefore, HHUA cells underwent apoptotic cell death by exposure to anti-Fas mAb.
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Caspase activity in HHUA cells and human endometrial tissue during the menstrual cycle
HHUA cells were harvested at specified periods after exposure to 100 ng/ml anti-Fas mAb, and caspase activation by colourimetric assays was analysed. The activity levels of caspase-3, -8 and -9 in HHUA cells significantly increased 3–9 h after exposure to anti-Fas mAb, as compared with non-treated cells in the order shown in Figure 3a. Caspase-3 and -8 activities were low in human endometrial tissue from the early proliferative phase to the early secretory phase of the menstrual cycle and reached a significant peak during the late secretory phase (approximately four-fold more caspase-3 and approximately three-fold more caspase-8) (P < 0.01) among all phases of the menstrual cycle (Figure 3b). Caspase-9 was inactive from the early proliferative, to the early secretory, phase but active during the late secretory phase. The order of caspase activity levels in endometrial tissue was similar to that in HHUA cells exposed to anti-Fas mAb.
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Effect of caspase inhibitors on apoptosis of HHUA cells
We analysed the viability of HHUA cells that were incubated with caspase inhibitors before anti-Fas mAb. The specific caspase inhibitors were Z-VAD-FMK for pan-caspase, Ac-IETD-CHO for caspase-8, Ac-LEHD-CHO for caspase-9 and Ac-DEVD-CHO for caspase-3. Various concentrations of these caspase inhibitors did not affect the viability of the HHUA cells (data not shown). Prior exposure to Z-VAD-FMK rescued HHUA cells from anti-Fas-mAb-induced apoptosis in a dose-dependent manner. The cell viability after treatment with Ac-IETD-CHO, Ac-DEVD-CHO and Ac-LEHD-CHO was clearly lower than cells treated with Z-VAD-FMK, and all three inhibitors prevented apoptosis in HHUA cells only at relatively high concentrations (Figure 4).
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Cytochrome c release in HHUA cells and human endometrial tissue
Cytochrome c is released from the mitochondrial membrane into the cytosol to activate caspase-9 when apoptosis proceeds via the mitochondrial pathway. Cytochrome c was undetectable in the cytosolic fraction of HHUA cells before exposure to anti-Fas mAb (Figure 5a, lane N). However, cytochrome c was significantly elevated 3 h thereafter (Figure 5a, lanes 3–9) and increased gradually up to 9 h (Figure 5b). The cytosol of human endometrial tissue did not contain cytochrome c during the proliferative phase (Figure 5c). However, in endometrial tissue, the release of cytochrome c was detected from the early secretory phase (days 21 and 23) and was significantly elevated on days 25 and 27 of the menstrual cycle (Figure 5c and d).
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Bid expression in HHUA cells and human endometrial tissue
Full-length Bid that was present as a doublet in HHUA cells (Figure 5a) began to degrade in 6 h after anti-Fas mAb exposure (P < 0.05, Figure 5b) and disappeared 24 h thereafter (data not shown). In human endometrial tissue, the expression of full-length Bid was cyclic, peaking on day 16 and disappearing during the late secretory phase (P < 0.01, Figure 5c and d).
FLIP expression in HHUA cells and human endometrial tissue
The antibody used in this study recognizes both long and short forms of FLIP (FLIPL and FLIPS) (Irmler et al., 1997; Scaffidi et al., 1999). However, FLIPL identified as p55 was expressed in HHUA cells before exposure to anti-Fas mAb and decreased by 6 h and beyond (P < 0.05, Figure 6a and b). On the other hand, the FLIPS level identified as p31 did not decrease (Figure 6a and b) and remained at the same level 24 h after antibody exposure (data not shown). Expression of p29 significantly increased 3 h and beyond after antibody exposure (P < 0.01, Figure 6a and b). The protein p37.5 was consistently expressed 0–9 h after antibody exposure (Figure 6a). In human endometrial tissue, only FLIPS was detected and decreased slightly, but significantly, at the late secretory phase (P < 0.05, Figure 6c and d).
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Discussion |
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It is well known that there are the two major apoptotic pathways in mammalian cells i.e., the death receptor pathway and the mitochondrial pathway. Why these two pathways exist and which pathway is dominant in mammalian cells remains to be determined. When FasL binds to Fas in the death receptor pathway, FADD and procaspase-8 are recruited to the intracellular domain (DD) of Fas where they form a complex called DISC (Kischkel et al., 1995
). Procaspase-8 is activated in DISC by autoprocessing, and active caspase-8 cleaves caspase-3 and other molecules such as Bid. The carboxyl fragment of Bid acts as an input signal to the mitochondria (Luo et al., 1998) and is transported from the cytosol to the mitochondria where it induces the translocation of cytochrome c. Next, cytochrome c associated with Apaf-1, and procaspase-9 activates caspase-9 and -3 (Hengartner, 2000)]. Therefore, activated caspase-8 can switch on both the death receptor pathway and the mitochondrial pathway via Bid degradation.
Our previous in vivo studies (Otsuki et al., 1994; Yamashita et al., 1999; Otsuki, 2001) demonstrated that human endometrial cell apoptosis involves the Bcl-2, Fas/FasL system and the caspase family. This present study clearly shows the increased caspase-3, -8 and -9 activity, Bid degradation and cytochrome c translocation from the mitochondria membrane into the cytosol of human endometrial cells during the late secretory phase. Therefore, these findings indicate both the mitochondrial and death receptor pathways are involved in apoptosis of human endometrial cells, but which of the death receptor or mitochondrial pathways is activated in human endometrial cells is unknown. HHUA cells were used to further investigate human endometrial apoptotic signalling. Fas and FasL are co-expressed in human endometrial epithelial cells throughout the menstrual cycle (Yamashita et al., 1999). HHUA cells were reactive to estrogen (Figure 1a), and the results were in agreement with a previous report (Ishiwata et al., 1984a). However, HHUA cells did not express ER, instead ERß was expressed (Figure 1b). The reason why E2 treatment enhanced the cell proliferation may be due to the ERß pathway. Nevertheless, the HHUA cell line maintains the endometrial epithelial cell characteristics during the secretory phase (Ishiwata et al., 1984a). Furthermore, this cell line expresses Fas on the cell membrane. Therefore, HHUA cells are considered useful for in vitro studies of apoptotic signalling in the human endometrium. In the present study, the proliferation of HHUA cells was suppressed by treatment with progesterone without PR expression (Figure 1b), but apoptosis did not occur in agreement with previous reports (Ishiwata et al., 1984a; Figure 1A). However, treatment with progesterone may cause some cytotoxic effects to HHUA cells. When anti-Fas mAb induces HHUA cell apoptosis, caspase-3, -8, and -9 activities increase, cytochrome c is released into the cytosol and Bid is degraded completely, as seen in human endometrial cells. The caspase inhibitor study using HHUA cells exposed to anti-Fas mAb demonstrated that caspase-9 inhibition yielded the same effect of blocking apoptosis as caspase-8. This result showed that the mitochondrial pathway via Bid degradation dominates in the Fas-mediated apoptosis of HHUA cells. Therefore, the apoptotic signalling in HHUA cells may explain human endometrial apoptosis; in that, apoptosis is induced by the mitochondrial pathway via Bid degradation during the secretory phase in the human endometrium.
However, it is not clear why apoptotic endometrial cells first appear from the late secretory phase to the menstrual phase and not from the early secretory phase when the Bcl-2 expression level abruptly decreases in human endometrial cells. Lymphoid cells such as Jurkat cells incubated with anti-Fas mAb usually undergo apoptosis within 1h. HHUA cells became apoptotic from 3 to 9 h after exposure to anti-Fas mAb, which is later than Jurkat cells. These findings indicate that an anti-apoptotic molecule other than Bcl-2 is involved in the apoptotic signal pathway for both human endometrial and HHUA cells.
FLIP is an anti-apoptotic molecule of the death receptor pathway (Irmler et al., 1997), and HHUA cells express the splice variants, FLIPL and FLIPS. Furthermore, the 37.5 kDa protein (p37.5) reacts with anti-FLIP mAb. As we detected this band in the presence of another anti-FLIP antibody, the band is specific. When incorporated into the DISC complex, FLIP inhibits procaspase-8 autoprocessing (Scaffidi et al., 1999). At this point, FLIPL itself is processed by procaspase-8, and the processed form is called p43. Thus, p37.5 detected in HHUA cells may be related to p43 in some way. A 29 kDa protein appeared in HHUA cells treated with anti-Fas mAb. Although p29 may be a cleavage protein from FLIPL, it is unclear whether p29 is functional. Further investigation is thus necessary. Between 3 and 6 h after treatment with anti-Fas mAb, the FLIPL expression level in HHUA cells decreased and was inversely proportional to the activity levels of caspase-3, -8 and -9. These findings indicate that FLIP in HHUA cells inhibit caspase-8 a few hours after exposure to anti-Fas mAb and that 6 h after antibody exposure, HHUA cells may undergo apoptosis due to FLIP consumption. On the other hand, FLIPS were only detected in human endometrial tissue during the menstrual cycle. Although FLIPS was ubiquitously expressed in normal endometrial tissue, since FLIPS expression was slightly but significantly decreased during the late secretory phase in normal endometrial tissue, it is possible that FLIPS may regulate menstruation in normal human endometrial tissue; however, further investigation is required. Furthermore, FLIPL in HHUA cells may in fact block apoptosis via the Fas/FasL system.
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Acknowledgements |
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We gratefully acknowledge the surgeons at the Takatsuki Red Cross Hospital (Osaka, Japan) and the Hirakata City Hospital (Osaka, Japan). This work was supported in part by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (No. 14571592 to Y.O. and No. 14770873 to H.A.).
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Submitted on November 8, 2005; resubmitted on November 30, 2005; accepted on December 7, 2005.
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