Tracy A. Christianson

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-γ/TNF-α-mediated cytotoxicity

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α) and double‐stranded RNA (dsRNA). Because apoptotic responses of mutant FA‐C cells involve activation of interferon‐inducible, dsRNA‐dependent protein kinase PKR, we sought to identify FANCC‐binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using ‘pull‐down’ and co‐immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70–FANCC binding and protection from IFN‐γ/TNF‐α‐induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70‐interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN‐γ and TNF‐α.

The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors.

ABSTRACT Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-γ) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-γ receptor α chain (IFN-γRα) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-γR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-γ-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-γ hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1−/− mice were resistant to IFN-γ. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT−/− mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).

The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded RNA-dependent protein kinase-mediated death signaling pathway requires the Fanconi anemia protein, FANCC.

Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents. A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR). Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex. By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR bothin vivo and in vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins. Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR. We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function.

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice

To define the mechanism of cyclosporine (CsA)‐induced apoptosis, we investigated the expression of apoptosis‐related genes in experimental chronic CsA nephrotoxicity. Mice on a low‐salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) stain, and the expressions of apoptosis‐related genes were evaluated by reverse transcription‐polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 ± 41 vs. 3 ± 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas‐ligand mRNA (0.20 vs. 0.02 amol/μg total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/μg total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 ρmol/μg/ h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 ρmol/μg/ h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl‐2 protein increased significantly in the CsA group (5.3‐fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas‐ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis‐related genes is associated with CsA‐induced apoptotic cell death.

SKOV3 ovarian carcinoma cells have functional estrogen receptor but are growth-resistant to estrogen and antiestrogens

Estrogen receptor positive ovarian cancer is often refractile to antiestrogen therapy. Here we describe the SKOV3 human ovarian carcinoma cell line as an in vitro model for estrogen and antiestrogen resistant ovarian cancer. While SKOV3 cells expressed estrogen receptor (ER) mRNA and protein at a similar level as the estrogen responsive T47D breast carcinoma cell line, their growth was not responsive to estradiol (E2) and was not inhibited by the antiestrogens OH-tamoxifen and ICI 164,384. The ER in SKOV3 cells was normal with respect to apparent Kd for binding with E2, E2 regulation of a transiently transfected ERE driven reporter gene, and E2 stimulation of expression of the early growth response genes c-myc and c-fos. However, the SKOV3 cells exhibited no expression of the progesterone receptor gene (PR) even after addition of E2, and the protein products of the estrogen responsive genes HER-2/neu and cathepsin D were expressed at constitutive levels that were not regulated by E2. Therefore, estrogen resistance in these cells may be a result of constitutive expression and loss of E2 regulation of selected growth regulatory gene products rather than a defect in estrogen activation of ER as a transcriptional regulator.

Structure-function analysis of the fanconi anemia complementation group c (Fancc) Gene reveals different requirements for STAT-1 activation and for resistance to cross-linking agents

Abstract The FA group C gene product (FANCC) is required for resisting alkylating agents and for optimal activation of Stat-1 in normal cytokine-stimulated cells. Seeking to isolate structural determinants of these two functions of FANCC, we transduced cells obtained from children with FA-C with retroviral vectors expressing six alanine mutants of FANCC cDNA and tested cell survival in mitomycin C (MMC) and Stat-1 activation in response to IFNγ. All mutants effectively complemented the alkylator hypersensitive phenotype, but two mutations, S249A and E251A, failed to correct defective Stat-1 activation in the FA-C lymphoblast cell line HSC536N. We next asked whether this domain might be functionally preserved in some milder cases of FA. In the FA-C cell line PD4 which expresses an N-terminally truncated mutant 55 kDa protein (M55) with normal S249 and E251, we determined that Stat-1 is normally phosphorylated. Moreover, M55 cDNA complemented the Stat-1 activation defect in HSC536 cells without reversing MMC sensitivity. Thus, a central domain of the FANCC protein is required for functional interaction of FANCC with Stat-1 and differs from structural elements required for MMC protection which do not tolerate N-terminal truncation. Functional preservation of this domain may explain the less severe hematologic phenotype in selected children with FA-C.

Fanconi anemia and apoptosis: The fancc gene product functions downstream of caspase 3

Abstract Hematopoietic progenitor cells (HPC) from mice and children with type C Fanconi anemia (FA-C) are hypersensitive to mitotic inhibitory factors, including interferon-γ (IFN-γ) and tumor necrosis factor-α (TNFα)(Blood, 90:974,1997). We tested the hypothesis that IFN/fasL induced programmed cell death in FA-C cells involves the ordered activation of specific caspase molecules. In immunoblot experiments, the FA-C lymphoblastoid cell line HSC536N treated with an agonistic fas antibody (100 ng/ml) and IFN-γ(1 ng/ml) contained activated caspase 3, 6, and 7 (but not 1, 2, 4, or 10) by 60 minutes and PARP cleavage products by 180 minutes. Caspase 3 activation by immunoblot and flow cytometric analysis was identical in FA-C cells and FA cells complemented by FANCC cDNA transfer even though the latter cells were less apoptotic. The apoptotic effects of fas agonists in IFN-γ-treated human FA-C CD34 + cells, FA-C murine progenitor cells, and EBV-transformed lymphoblasts from a child with FA of the C type, were blocked when the cells were pretreated any one of three inhibitors of caspase 3 protease. Caspase 1 inhibitors did not block the effect of IFN and fas ligand and caspase 1 inhibition did not prevent caspase 3 cleavage but caspase 8 inhibition did block caspase 3 activation. We conclude that in IFN-primed FA-C cells, fas -induced apoptosis involves the activation of caspase 8, which controls activation of caspase 3 family members but that the function of FANCC in suppressing normal apoptotic responses is downstream of caspase 3.