A novel cancer risk prediction score for the natural course of FA patients with biallelic BRCA2/FANCD1 mutations.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7 years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3 years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3 years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0 years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2.

Identification of new RAD51D-regulating microRNAs that also emerge as potent inhibitors of the Fanconi anemia/homologous recombination pathways.

The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. First, to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germline mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in green fluorescent protein (GFP) repair assays. In summary, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with biallelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.

Spotlight on CYP4B1.

The mammalian cytochrome P450 monooxygenase CYP4B1 can bioactivate a wide range of xenobiotics, such as its defining/hallmark substrate 4-ipomeanol leading to tissue-specific toxicities. Similar to other members of the CYP4 family, CYP4B1 has the ability to hydroxylate fatty acids and fatty alcohols. Structural insights into the enigmatic role of CYP4B1 with functions in both, xenobiotic and endobiotic metabolism, as well as its unusual heme-binding characteristics are now possible by the recently solved crystal structures of native rabbit CYP4B1 and the p.E310A variant. Importantly, CYP4B1 does not play a major role in hepatic P450-catalyzed phase I drug metabolism due to its predominant extra-hepatic expression, mainly in the lung. In addition, no catalytic activity of human CYP4B1 has been observed owing to a unique substitution of an evolutionary strongly conserved proline 427 to serine. Nevertheless, association of CYP4B1 expression patterns with various cancers and potential roles in cancer development have been reported for the human enzyme. This review will summarize the current status of CYP4B1 research with a spotlight on its roles in the metabolism of endogenous and exogenous compounds, structural properties, and cancer association, as well as its potential application in suicide gene approaches for targeted cancer therapy.

Synergistic Interaction of the Class IIa HDAC Inhibitor CHDI0039 with Bortezomib in Head and Neck Cancer Cells.

In contrast to class I/IIb/pan histone deacetylase inhibitors (HDACi), the role of class IIa HDACi as anti-cancer chemosensitizing agents is less well understood. Here, we studied the effects of HDAC4 in particular and the class IIa HDACi CHDI0039 on proliferation and chemosensitivity in Cal27 and cisplatin-resistant Cal27CisR head and neck squamous cell cancer (HNSCC). HDAC4 and HDAC5 overexpression clones were generated. HDAC4 overexpression (Cal27_HDAC4) increased proliferation significantly compared to vector control cells (Cal27_VC). Chicken chorioallantoic membrane (CAM) studies confirmed the in vitro results: Cal27_HDAC4 tumors were slightly larger than tumors from Cal27_VC, and treatment with CHDI0039 resulted in a significant decrease in tumor size and weight of Cal27_HDAC4 but not Cal27_VC. Unlike class I/pan-HDACi, treatment with CHDI0039 had only a marginal impact on cisplatin cytotoxicity irrespective of HDAC4 and HDAC5 expression. In contrast, the combination of CHDI0039 with bortezomib was synergistic (Chou-Talalay) in MTT and caspase 3/7 activation experiments. RNAseq indicated that treatment with CHDI0039 alters the expression of genes whose up- or downregulation is associated with increased survival in HNSCC patients according to Kaplan-Meier data. We conclude that the combination of class IIa HDACi with proteasome inhibitors constitutes an effective treatment option for HNSCC, particularly for platinum-resistant cancers.

Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.

Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People.

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1.

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, ß-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.

Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4-Ipomeanol.

A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

Upregulation of the long non-coding RNA CASC9 as a biomarker for squamous cell carcinoma.

BACKGROUND: Few diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC. METHODS: Public datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines. RESULTS: Data mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity. CONCLUSIONS: CASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor.

Structure-Activity Relationships for CYP4B1 Bioactivation of 4-Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates.

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2-but not FANCI-plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency.

UNLABELLED: Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. CONCLUSIONS: PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.

The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling.

BACKGROUND: Renal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain). METHODS: Expression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student's t-test. RESULTS: Regarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells. CONCLUSION: Taken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene.

BACKGROUND: Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. OBJECTIVE: Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. METHODS: Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. RESULTS: Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. CONCLUSIONS: XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.

Peptide-independent stabilization of MHC class I molecules breaches cellular quality control.

The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K(b)-Y84C, in which two α-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. K(b)-Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (ß2m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and ß2m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by ß2m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that ß2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.

Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

A natural tapasin isoform lacking exon 3 modifies peptide loading complex function.

To assure efficient MHC class I (MHC-I) peptide loading, the peptide loading complex (PLC) recruits the peptide-receptive form of MHC-I, and in this process, tapasin (tpn) connects MHC-I with the peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G-runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)-family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys-95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC-I in tpn-deficient cells, in tpn-proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn-dependent HLA-B*44:02 allele; the stability of the tpn-independent HLA-B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC-I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8(+) T-cell responses.

Characterization and manipulation of foamy virus membrane interactions.

Foamy viruses (FVs), a unique type of retroviruses, are characterized by several unusual features in their replication strategy. FVs, common to all non-human primates and several other species, display an extremely broad tropism in vitro. Basically, all mammalian cells and species examined, but also cells of amphibian or bird origin, are permissive to FV glycoprotein (Env)-mediated capsid release into the cytoplasm. The nature of the broadly expressed, and potentially evolutionary conserved, FV entry receptor molecule(s) is poorly characterized. Although recent data indicate that proteoglycans serve as an important factor for FV Env-mediated target cell attachment, additional uncharacterized molecules appear to be essential for the pH-dependent fusion of viral and cellular lipid membranes after endocytic uptake of virions. Furthermore, FVs show a very special assembly strategy. Unlike other retroviruses, the FV capsid precursor protein (Gag) undergoes only very limited proteolytic processing during assembly. This results in an immature morphology of capsids found in released FV virions. In addition, the FV Gag protein appears to lack a functional membrane-targeting signal. As a consequence, FVs utilize a specific interaction between capsid and cognate viral glycoprotein for initiation of thebudding process. Genetic fusion of heterologous targeting domains for plasma but not endosomal membranes to FV Gag enables glycoprotein-independent particle egress. However, this is at the expense of normal capsid morphogenesis and infectivity. The low-level Gag precursor processing and the requirement for a reversible, artificial Gag membrane association for effective pseudotyping of FV capsids by heterologous glycoproteins strongly suggest that FVs require a transient interaction of capsids with cellular membranes for viral replication. Under natural condition, this appears to be achieved by the lack of a membrane-targeting function of the FV Gag protein and the accomplishment of capsid membrane attachment through an unusual specific interaction with the cognate glycoprotein.