Fas-associated Phosphatase-1 Research Articles

Tumor necrosis factor p55 and p75 receptors are involved in chemical‐induced apoptosis of dentate granule neurons

Localized tumor necrosis factor-alpha (TNFalpha) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFalpha mRNA levels. TNFp55R (55 kDa type-1 TNFR) and TNFp75R (75 kDa type-2 TNFR) immunoreactivity in AC3(+) neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFalpha antibody and in TNFp55R-/-p75R-/- mice that showed higher constitutive mRNA levels for interleukin (IL-1alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), TNFalpha, transforming growth factor beta1, Fas, and TNFRSF6-assoicated via death domain (FADD). TNFp75R-/- mice showed exacerbated injury and elevated mRNA levels for IL-1alpha, MIP-1alpha, and TNFalpha. In TNFp55R-/- mice, constitutive mRNA levels for TNFalpha, IL-6, caspase 8, FADD, and Fas-associated phosphatase were higher; IL-1alpha, MIP-1alpha, and transforming growth factor beta1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co-expression of TNFp75R in AC3(+) neurons. The data demonstrate TNFR-mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R-mediated apoptosis in the absence of normal TNFp55R activity.

The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Expression and localization of Fas-associated proteins following focal cerebral ischemia in rats

The aim of this study was to investigate the changes of expression of Fas-associated proteins and its cellular localization in the peri-infarct region following transient focal cerebral ischemia. Adult male Sprague–Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 2 h and reperfusion for 1, 3, 6, 12 and 24 h. The expression of Fas-associated death domain protein (FADD), Fas-associated phosphatase-1 (FAP-1) caspase-8 and death-associated protein (Daxx), the pro-apoptotic genes, were examined by methods of RT-PCR, immunohistochemistry and Western blot. The results showed that the expression levels of mRNA and protein for FADD and caspase-8 increased significantly at 1–3 h after reperfusion, peaked at 12 h, then declined markedly at 24 h. The time course change of FAP-1 was consistent with that of FADD. The expression level of mRNA and protein for death-associated protein (Daxx) increased significantly at 3 h after reperfusion and persisted for 24 h at a high level. Immunofluorescence double-staining laser scanning showed that the immunoreactivity of FADD was localized in cytoplasm, and Daxx immunoreactivity was translocated from nucleus to cytoplasm at 3 h after reperfusion. The TUNEL-positive cells could be found in peri-infarct region at 3 h and increased with time after reperfusion. Our findings suggest a possible association between expression of FADD, caspase-8, Daxx and FAP-1 genes and apoptosis following ischemia.

CCM3 interacts with CCM2 indicating common pathogenesis for cerebral cavernous malformations

Individuals carrying a mutation in one of the three cerebral cavernous malformation genes (CCM1/KRIT1, CCM2, CCM3) cannot be clinically distinguished, raising the possibility that they act within common molecular pathways. In this study, we demonstrate that CCM3 (PDCD10) coprecipitates and colocalizes with CCM2. We also show that CCM3 directly binds to serine/threonine kinase 25 (STK25, YSK1, SOK1) and the phosphatase domain of Fas-associated phosphatase-1 (FAP-1, PTPN13, PTP-Bas, PTP-BL). CCM3 is phosphorylated by STK25 but not by its other Yeast-Two hybrid interactor STK24, whereas the C-terminal catalytic domain of FAP-1 dephosphorylates CCM3. Finally, our experiments reveal that STK25 forms a protein complex with CCM2. Thus, our data link two proteins of unknown function, CCM3 and STK25, with CCM2, which is part of signaling pathways essential for vascular development and CCM pathogenesis.

FAP‐1‐mediated activation of NF‐κB induces resistance of head and neck cancer to fas‐induced apoptosis

Molecular mechanisms responsible for tumor resistance to apoptosis often involve the Fas/FasL pathway. While squamous cell carcinomas of the head and neck (SCCHN) express both Fas and FasL, their resistance to self-induced apoptosis or apoptosis mediated by Fas agonistic antibody (CH-11Ab) was independent of the level of Fas surface expression or the presence of soluble Fas in supernatants of primary or metastatic SCCHN cell lines. By in vitro immunoselection, using PCI-15A cell line treated with successive cycles of CH-11 Ab, Fas-resistant sublines with the parental genotype were selected. Such sublines failed to cleave caspase-8 upon Fas engagement and were resistant to CH-11 Ab, although they remained sensitive to VP-16 or staurosporin. In the presence of cycloheximide, the selected SCCHN sublines become susceptible to CH-11 Ab, and showed cleavage of caspase-8, suggesting that apoptosis resistance was mediated by an inhibitory protein(s) acting upstream of caspase-8. Overexpression of Fas-associated phosphatase 1 (FAP-1), but not cellular FLICE-inhibitory protein (cFLIP) in SCCHN sublines was documented by Western blots and RT-PCR analyses. The FAP-1+ selected sublines also downregulated cell surface Fas. A high phosphorylation level of IkappaB kappa, NFkappaB activation and upregulation of Bcl-2 expression were observed in the FAP-1+ sublines. Treatment with the phosphatase inhibitor, orthovanadate, or silencing of FAP-1 with siRNA abolished their resistance to apoptosis, suggesting that FAP-1 phosphatase activity could be responsible for NF-kappaB activation and resistance of SCCHN cells to Fas-mediated apoptosis.

Ionizing radiation suppresses FAP-1 mRNA level in A549 cells via p53 activation

Ionizing radiation (IR) is known to upregulate cell surface Fas through p53 activation in various cells. However, the signaling pathway intermediating between p53 activation and cell surface Fas upregulation remains to be elucidated. Recently, Fas-associated phosphatase-1 (FAP-1) has been reported to associate with Fas and inhibit cell surface Fas expression. We evaluated the expression of FAP-1 mRNA following IR in A549 cells. Ionizing radiation inhibited the expression of FAP-1 mRNA. Pretreatment with p53 inhibitor pifithrin α cancelled the IR-induced downregulation of FAP-1 mRNA. These results suggest that IR-induced p53 activation may upregulate cell surface Fas via the down-modulation of FAP-1.

Genetic characterization of fas-associated phosphatase-1 as a putative tumor suppressor gene on chromosome 4q21.3 in hepatocellular carcinoma.

Allelic loss at chromosome 4q21-23 occurs frequently in human hepatocellular carcinoma, and the putative tumor suppressor gene (TSG) has not yet been identified. We studied the Fas-associated phosphatase-1 (FAP-1) gene as a potential candidate TSG in this region. The expression level of FAP-1 RNA in hepatocellular carcinomas was evaluated by RNase protection and quantitative PCR. Sodium bisulfite modification and subsequent single-strand conformational polymorphism and sequence analyses were used to assay the methylation of CpGs at FAP-1 promoter. Direct sequencing of the FAP-1 coding region was conducted for detecting the genetic mutations. Two common single nucleotide polymorphisms of FAP-1 were selected for evaluating their association with the hepatocellular carcinoma trait in sporadic and familial hepatocellular carcinomas. Moreover, the functional effect of FAP-1 on cellular proliferation has been evaluated by small interfering RNA approach. Around 50% of hepatocellular carcinomas showed significantly decreased expression of FAP-1 compared with the corresponding nontumorous liver tissues. In most cases, the RNA level was well correlated with the methylation status of promoter CpGs, suggesting that the promoter methylation may contribute to the down-regulation. Several genetic mutations of FAP-1 have been identified in hepatocellular carcinomas. The G/G genotype of FAP-1 cSNP6304 was significantly associated with the increased risk of multiplex familial hepatocellular carcinomas (odds ratio, 2.44; 95% confidence interval, 1.19-5.01). Finally, knockdown expression of FAP-1 was shown to enhance the cellular proliferation in PLC5 cells. FAP-1 could be inactivated during hepatocarcinogenesis, mainly attributed by allelic loss and promoter methylation. The genetic mutations and polymorphisms may also confront with the higher hepatocellular carcinoma risk. These results first suggested FAP-1 as a putative TSG in hepatocarcinogenesis.

Fas‐associated phosphatase‐1 promotes Fas‐mediated apoptosis in human colon cancer cells: Novel function of FAP‐1

Fas-associated phosphatase-1 (FAP-1) has been thought as an inhibitor in Fas-mediated apoptosis. Here, we investigated the role of FAP-1 in Fas-mediated apoptosis of human colon cancer cells. The viability of four colon cancer cell lines treated with agonistic anti-Fas antibody was determined using WST-1 assay and cell death detection ELISA. pRc/CMV-FAP-1 was transfected to a FAP-1-negative, Fas-resistant colon cancer cell line SW480 by lipofection and the clones expressing FAP-1 protein were selected by limiting dilution. In the clones, expression of 550 genes was analyzed by cDNA microarrays. Protein expression of FAP-1 and molecules related to apoptosis was examined by western blot. We obtained two FAP-1 overexpressed clones which were much more susceptible to Fas-mediated apoptosis than control cells. In the clones, caspase 8 and caspase 3 were fully activated by agonistic anti-Fas antibody treatment. Bcl-2 family proteins were not related to the high susceptibility of these clones, because caspase 9 was not activated. Transfection of FAP-1 did not suppress the survival actions of insulin-like growth factor (IGF-1) which enhanced survival signal through Akt phosphorylation. Upregulation in 21 genes and downregulation in 29 genes was revealed by cDNA arrays. We confirmed protein expression of p21 and phosphorylated p21 were much more enhanced in the clones than in control cells. Overexpression of FAP-1 enhanced susceptibility to Fas-mediated apoptosis in SW480 and upregulation of p21 may contribute to this phenomenon. Our results indicate a novel function of FAP-1 in Fas-mediated apoptosis of human colon cancer cells.

FAS Associated Phosphatase (FAP-1) Blocks Apoptosis of Astrocytomas through Dephosphorylation of FAS

Astrocytomas are the most common primary tumor of the adult human central nervous system. Despite efforts to develop more effective clinical treatment strategies, median survival time for patients with the most severe form of astrocytoma, glioblastoma multiforme (GBM), remains about one year. Astrocytomas are resistant to cytotoxic therapy in general and radiation therapy in particular, greatly limiting treatment options. One reason for this seems to be defects in the pathways controlling apoptosis. We have characterized the role of the tyrosine phosphatase FAP-1 (FAS-associated phosphatase 1) in astrocytomas. Our studies demonstrate that FAP-1 is overexpressed in astrocytomas and this contributes to the resistance of the tumor cells to FAS-mediated apoptosis. We demonstrate that knockdown of FAP-1 by RNA interference leads to increased apoptosis and increased sensitivity of astrocytoma cells to FAS-induced cell death. FAP-1 binds to FAS in a ligand-dependent manner and forms a signaling complex that modulates the ability of astrocytoma cells to undergo FAS ligand (FASL)-mediated cell death. In astrocytoma cells, FASL treatment induces tyrosine phosphorylation of FAS. FAP-1 dephosphorylates phospho-tyrosine 275 in the carboxyl terminus of FAS. This is the first direct evidence that FAS activity can be regulated by reversible phosphorylation and suggests a mechanism for astrocytoma resistance to apoptosis.

Fas-associated phosphatase–1 (FAP–1) in Alzheimer’s disease hippocampus

Fas-associated phosphatase–1 (FAP–1) in Alzheimer’s disease hippocampus

Fas Binding to Calmodulin Regulates Apoptosis in Osteoclasts

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.

Immunohistochemical Localization of Fas-Associated Phosphatase-1 (FAP-1) in Alzheimer Disease Hippocampus

Fas-associated phosphatase-1 (FAP-1) is a regulatory peptide inhibiting apoptotic signal transduction via the death receptor Fas. Because apoptosis is a common mechanism leading to neuronal death in neurodegenerative disorders, the authors investigated the immunohistochemical distribution of FAP-1 in the hippocampus of elderly control subjects and Alzheimer disease (AD) patients. The current study provides the first evidence that FAP-1 is localized in the human hippocampus in pyramidal neurons of the hippocampal subfields CA1-4 and in granular cells. Cellular and extracellular FAP-1 intensity was increased in some control subjects and AD patients, but was not related to the stage of the illness. Rather, these data may indicate a general role for FAP-1 in neuronal death both in adult CNS and during the course of neurodegenerative disorders.

Novel role of CD40 in Fas‐dependent apoptosis of cultured salivary epithelial cells from patients with Sjögren's syndrome

To determine the role of Fas and CD40 in the molecular mechanism of salivary epithelial cell death in Sjogren's syndrome (SS). The expression of Fas and CD40 in SS salivary epithelial cells was analyzed by flow cytometry. Induction of apoptosis with anti-Fas and/or anti-CD40 monoclonal antibodies (mAb) was examined by morphologic analysis, DNA fragmentation, and TUNEL assay. Expression of c-FLIP, Fas-associated phosphatase 1, FADD, Bcl-2, Bcl-x(L), and Bcl-x(S) was determined by Western blot analysis. Expression of Fas and CD40 was significantly higher in SS salivary epithelial cells than in normal cells after interferon-gamma (IFNgamma) stimulation (P < 0.001 for both Fas and CD40). Although neither anti-Fas (CH11) nor anti-CD40 mAb alone could induce typical apoptosis, the two together and preincubation with IFNgamma efficiently induced apoptosis in SS salivary epithelial cells. This apoptosis was almost completely blocked by neutralizing anti-Fas mAb (ZB4), whereas an antagonistic mAb to CD40 (ch5D12) partially inhibited anti-Fas/anti-CD40-induced apoptosis. Also, c-FLIP, an important inhibitory molecule in the Fas death pathway, was strongly expressed in SS salivary epithelial cells, but its expression was down-regulated at the protein level by anti-CD40 mAb. CD40 signals promote Fas-dependent death of SS salivary epithelial cells by down-regulating c-FLIP expression. The presence of c-FLIP in these cells may explain their resistance to undergoing apoptosis in response to either anti-Fas or anti-CD40 mAb, despite their surface expression of both proteins. These findings suggest that SS salivary epithelial cell death requires the cooperation of both Fas and CD40.

Expression of FAP-1 by human colon adenocarcinoma: implication for resistance against Fas-mediated apoptosis in cancer

Although colon carcinoma cells express Fas receptors, they are resistant to Fas-mediated apoptosis. Defects within the intracellular Fas signal transduction may be responsible. We investigated whether the Fas-associated phosphatase-1 (FAP-1), an inhibitor of Fas signal transduction, contributed to this resistance in colon carcinomas. In vivo, apoptosis of cancer cells was detected in situ using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL). FAP-1, FasR, and Fas ligand (FasL) were detected using immunohistochemistry. In vitro, colon carcinoma cells were primarily cultured, and their sensitivity to Fas-mediated apoptosis was evaluated by treatment with agonistic anti-FasR CH11 IgM monoclonal antibody in the presence or absence of synthetic Ac-SLV (serine-leucine-valine) tripeptide. Fas-associated phosphatase-1 expression was detected in 20 out of 28 colon adenocarcinomas. In vivo, a positive correlation between the percentage of apoptotic tumour cells and the number of FasL-positive tumour infiltrating lymphocytes was observed in FAP-1 negative cancers, but not in FAP-1-positive ones. Primarily cultured colon cancer cells, which were refractory to CH-11-induced apoptosis, had higher expression of FAP-1 on protein and mRNA levels than the sensitive group. Resistance to Fas-mediated apoptosis in tumour cells could be abolished by Ac-SLV tripetides. Fas-associated phosphatase-1 expression protects colon cancer cells from Fas-mediated apoptosis, and blockade of FAP-1 and FasR interaction sensitises tumour cells to Fas-dependent apoptosis.

FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface.

As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression.

Head-to-head juxtaposition of Fas-associated phosphatase-1 (FAP-1) and c-Jun NH2-terminal kinase 3 (JNK3) genes: genomic structure and seven polymorphisms of the FAP-1 gene.

When characterizing the 5' flanking region of the c-Jun NH2-terminal kinase 3 ( JNK3) gene at 4q21-22, where frequent allelic losses and loss of expression had been detected in patients with brain tumors and hepatocellular carcinomas, we discovered that the Fas-associated phosphatase-1 ( FAP-1) gene was located only 633 bp upstream from JNK3 in a head-to-head orientation. A short G/C-rich region between the cap sites of the two genes suggested that they might share a bidirectional promoter region that appeared to contain multiple cis elements, including Sp1, AP-1, AP-2, GATA-1, a GC box, and a CCAAT box. The FAP-1 gene, consisting of 48 exons, initiates transcription within exon 2 and terminates in exon 48. Exons 2-5, 21-23, 25-28, 29-30, 33-34, and 34-36 encode six Gly-Leu-Gly-Phe repeat domains, and exons 12-17 and 44-88 encode the membrane-binding and catalytic domains, respectively. Seven polymorphisms were identified within functional domains or the putative promoter region, including two with amino acid substitutions, Leu1419Pro and Ile1522Met.

Modulation of death receptor-mediated apoptosis in differentiating human myeloid leukemia HL-60 cells

Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor alpha; (TNF-alpha), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-alpha-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-alpha sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-alpha-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.

Expression and Potential Role of Fas-Associated Phosphatase-1 in Ovarian Cancer

Fas-associated phosphatase-1 (FAP-1) is a protein-tyrosine phosphatase that binds the cytosolic tail of Fas (Apo1, CD95), presumably regulating Fas-induced apoptosis. Elevations of FAP-1 protein levels in some tumor cell lines have been correlated with resistance to Fas-induced apoptosis. To explore the expression of FAP-1 in ovarian cancer cell lines and archival tumor specimens, mouse monoclonal and rabbit polyclonal antibodies were generated against a FAP-1 peptide and recombinant FAP-1 protein. These antibodies were used for immunoblotting, immunohistochemistry, and flow-cytometry analysis of FAP-1 expression in the Fas-sensitive ovarian cancer lines HEY and BG-1, and in the Fas-resistant lines OVCAR-3 FR and SK-OV-3. All methods demonstrated high levels of FAP-1 in the resistant lines OVCAR-3 FR and SK-OV-3, but not in the Fas-sensitive lines HEY and BG-1. Furthermore, levels of FAP-1 protein also correlated with the amounts of FAP-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction analysis. FAP-1 protein levels were investigated by immunoblotting in the National Cancer Institute's panel of 60 human tumor cell lines. Although FAP-1 failed to correlate with Fas-resistance across the entire tumor panel, Fas-resistance correlated significantly with FAP-1 expression (P: < or = 0.05) and a low Fas/FAP-1 ratio (P: < or = 0.028) in ovarian cancer cell lines. FAP-1 expression was also evaluated in 95 archival ovarian cancer specimens using tissue-microarray technology. FAP-1 was expressed in nearly all tumors, regardless of histological type or grade, stage, patient age, response to chemotherapy, or patient survival. We conclude that FAP-1 correlates significantly with Fas resistance in ovarian cancer cell lines and is commonly expressed in ovarian cancers.

Pro-apoptotic and anti-apoptotic effects of transferrin and transferrin-derived glycans on hematopoietic cells and lymphocytes.

The aim of this study was to test the hypothesis that transferrin (Tf) has anti-apoptotic properties and thereby exerts a cytoprotective effect against tissue damage induced by irradiation and other cytotoxic modalities. This hypothesis was tested in several models, including in vitro human short-term marrow cultures, subpopulations of marrow cells, particularly, CD56(+) natural killer cells (and natural killer cell lines), and in vivo radioprotection of murine marrow cells. Reverse transcriptase polymerase chain reaction analysis was used for determination of cytokine mRNA. Preincubation of human marrow with Tf protected cells (except for a CD56(+) subpopulation) against cell death induced by gamma-irradiation, tumor necrosis factor-alpha (TNF-alpha), and agonistic anti-Fas monoclonal antibody. Deglycosylation of Tf abrogated this action of Tf; conversely, Tf-derived glycans (Tf-Gly) (but not glycans isolated from other proteins) mimicked the effects of the intact Tf molecule on apoptosis. Antibodies specific for the Tf receptor (CD71) did not block the effects of Tf or Tf-Gly on apoptosis. Determination of cytokine mRNA in the course of Fas-mediated apoptosis in the presence of Tf or Tf-Gly showed upregulation of mRNA for Fas ligand and TNF-alpha in CD56(+) and downregulation of these transcripts along with upregulation of mRNA for interleukin-10 in CD3(+) marrow cells. Under these conditions, a distinct increase in Fas-associated phosphatase-1 message was observed in CD3(+) cells that were protected by Tf or Tf-Gly against apoptosis. The in vitro data were confirmed in a murine in vivo model in which pretreatment of mice with Tf protected marrow cells against gamma-irradiation-induced cell death. These data suggest a role for Tf and particularly Tf-Gly in the regulation of programmed cell death, apparently via alterations in cytokine expression, and provide a basis for additional studies on the use of Tf in cytoprotective protocols.

Expression of Fas and Fas-related molecules in human hepatocellular carcinoma

Many tumor cells, including hepatocellular carcinoma (HCC), express both Fas and its ligand on their surfaces, and it has remained a mystery why such cells do not spontaneously become apoptotic. In the current study, we analyzed the alterations of Fas structure and the expression of Fas and Fas ligand (FasL) and of Fas pathway inhibitors, including soluble Fas (sFas), Fas-associated phosphatase-1 (FAP-1), and bcl-2, in 50 cases of human HCC. Monoallelic loss of the Fas gene, as determined by loss of heterozygosity with intragenic polymorphisms, was observed in 5 of the 34 informative cases (15%), but none of the 50 cases showed Fas gene mutation. Expression of Fas and FasL was detected in 44 (88%) and 50 (100%) cases, respectively. sFas messenger RNA, as analyzed by in situ reverse-transcription polymerase chain reaction was expressed in 42 of the 50 cases (84%), and FAP-1 expression was observed in 40 of the 50 cases (80%). In contrast, none of the 50 cases showed bcl-2 expression. Our results showed that the majority of the HCCs (88%) coexpressed a death receptor, Fas and its cognate ligand, FasL, but all HCCs showed one or more alterations of the Fas pathway molecules known to inhibit Fas-mediated apoptosis. These findings suggest that the expression of sFas and FAP-1 and, in part, loss of Fas expression, rather than Fas gene alteration or bcl-2 expression, may be involved in the Fas resistance of HCC in vivo and that these mechanisms may play important roles in the pathogenesis of human HCC. HUM PATHOL 32:250-256. Copyright © 2001 by W.B. Saunders Company