Crystal structures of human CD40 in complex with monoclonal antibodies dacetuzumab and bleselumab.

CD40 is a member of the tumor necrosis factor receptor superfamily, and it is widely expressed on immune and non-immune cell types. The interaction between CD40 and the CD40 ligand (CD40L) plays an essential function in signaling, and the CD40/CD40L complex works as an immune checkpoint molecule. CD40 has become a therapeutic target, and a variety of agonistic/antagonistic anti-CD40 monoclonal antibodies (mAbs) have been developed. To better understand the mode of action of anti-CD40 mAbs, we determined the X-ray crystal structures of dacetuzumab (agonist) and bleselumab (antagonist) in complex with the extracellular domain of human CD40, respectively. The structure reveals that dacetuzumab binds to CD40 on the top of cysteine-rich domain 1 (CRD1), which is the domain most distant from the cell surface, and it does not compete with CD40L binding. The binding interface of bleselumab spread between CRD2 and CRD1, overlapping with the binding surface of the ligand. Our results offer important insights for future structural and functional studies of CD40 and provide clues to understanding the mechanism of biological response. These data can be applied to developing new strategies for designing antibodies with more therapeutic efficacy.

Clinical significance of MUC13 in pancreatic ductal adenocarcinoma.

BACKGROUND: Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. METHODS: MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. RESULTS: MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS = 5.8; AUC >0.8, P < 0.0001), 94.6% of pancreatic ductal adenocarcinoma (PDAC) samples (MCS = 9.7, P < 0.0001) as compared to low expression in tumor adjacent tissues (MCS = 4, P < 0.001) along with faint or no expression in normal pancreatic tissues (MCS = 0.8; AUC >0.8; P < 0.0001). Nuclear MUC13 expression positively correlated with nodal metastasis (P < 0.05), invasion of cancer to peripheral tissues (P < 0.5) and poor patient survival (P < 0.05; prognostic AUC = 0.9). Submicron scale mass density and artificial intelligence based algorithm analyses also elucidated association of MUC13 with greater morphological disorder (P < 0.001) and nuclear MUC13 as strong predictor for cancer aggressiveness and poor patient survival. CONCLUSION: This study provides significant information regarding MUC13 expression/subcellular localization in PanCa samples and supporting the use anti-MUC13 MAb for the development of PanCa diagnostic/prognostic test.

Human Fc receptor-like 5 binds intact IgG via mechanisms distinct from those of Fc receptors.

Fc receptor-like (FCRL) 5 regulates B cell Ag receptor signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with ~1 µM KD, whereas the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab')2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense Ig quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced Abs.

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by reducing signaling activity through epitope selection.

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

Fc receptor-like 3 protein expressed on IL-2 nonresponsive subset of human regulatory T cells.

Fc receptor-like 3 (FCRL3) is a cell surface protein homologous to Fc receptors. The FCRL3 gene is present in humans but not in mice. We found that FCRL3 protein is expressed on 40% of human naturally occurring CD4(+) regulatory T (nTreg) cells (CD4(+)CD25(+)CD127(low)). Sorted nTreg cells with the surface phenotype FCRL3(+) and FCRL3(-) were both hypoproliferative to TCR stimulation and both suppressive on proliferation of conventional T cells (CD4(+)CD25(-)) in vitro. They both expressed forkhead box p3 (Foxp3) protein, the intracellular regulatory T cell marker. However, in contrast to FCRL3(-) nTreg cells, FCRL3(+) nTreg cells were not stimulated to proliferate by the addition of exogenous IL-2. In addition, Foxp3(+) cells induced from conventional T cells by TGF-beta treatment did not exhibit FCRL3 expression. These results suggest that the FCRL3(+) subset of human nTreg cells identified in this study arise in vivo and Foxp3 expression alone is not sufficient to induce FCRL3 expression. FCRL3 may be involved in human-specific mechanisms to control the generation of nTreg cells.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Expression of POTE protein in human testis detected by novel monoclonal antibodies.

The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

Volume-sensitive Cl(-) channel as a regulator of acquired cisplatin resistance.

The platinum-based drug cisplatin (cis-diamminedichloroplatinum (II)) is widely used in cancer therapy. However, cancer cells can develop resistance after exposure to cisplatin. Recently, many studies have pointed to the involvement of plasma membrane ion channels in a cell's response to cisplatin. Our group has found that pretreatment with cisplatin enhanced the activity of volume-sensitive C-channels in human epidermoid cancer KB cells; cisplatin-resistant KCP-4 cells derived from KB cells, on the other hand, lacked functional expression of these channels. This suggests that the activity of volume-sensitive Cl(-) channels is an important factor in determining the sensitivity of cancer cells to cisplatin. Furthermore, when volume-sensitive Cl(-) channel function was partially restored in cisplatin-resistant KCP-4 cells treated with a histone deacetylase inhibitor, KCP-4 cells exhibited a restoration of sensitivity to cisplatin; this increased sensitivity was inhibited by a volume-sensitive Cl(-) channel blocker. We therefore propose that impaired activity of the volume-sensitive Cl(-) channel is involved in the acquired cisplatin resistance of these cancer cells. In this review, we will outline the relationship between volume-sensitive Cl(-) channels, cisplatin-induced apoptosis, and cisplatin resistance. Activating the volume-sensitive outwardly-rectifying Cl(-) channel may be a new strategy in treating clinical cisplatin resistance.

Preclinical development of anti-BCMA immunotoxins targeting multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy that is incurable for the majority of patients. New treatments are urgently needed. Recombinant immunotoxins (RITs) are chimeric proteins that are composed of the Fv or Fab portion of an antibody fused to a bacterial toxin. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein and an ideal target for antibody-based treatments for MM. METHODS: RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in Escherichia coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays. We also measured their binding to human and mouse serum albumins and to BCMA and measured their serum half-life in mice. RESULTS: Using Fvs from different anti-BCMA antibodies, we produced RITs that specifically kill BCMA-expressing MM cells in vitro. To increase the serum half-life in vivo, we generated RITs that are fused with albumin-binding domains (ABDs). All RITs with ABDs have some decreased activity compared to the parent RIT, which is not due to decreased binding to BCMA. CONCLUSIONS: Various new anti-BCMA immunotoxins were produced and evaluated. None of these were better than LMB-75 (anti-BCMA BM306-disulfide-stabilized Fv-LRggs) supporting the further preclinical development of LMB-75.

Immunoglobulin superfamily receptor translocation associated 2 protein on lymphoma cell lines and hairy cell leukemia cells detected by novel monoclonal antibodies.

PURPOSE: The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of mRNA in B cell-lineage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. EXPERIMENTAL DESIGN: A mouse used for cell fusion was DNA-immunized with an expression plasmid encoding the IRTA2 cDNA. The reactivity of MAbs secreted from the hybridomas were characterized with recombinant proteins of IRTA family members in an enzyme immunoassay and a fluorescence-activated cell sorter (FACS). Nineteen human lymphoma cell lines and blood cells from five patients with hairy cell leukemia (HCL) were analyzed with IRTA2 expression using FACS. RESULTS: Three MAbs (F25, F56, and F119) were selected based on their specific reactivity with recombinant IRTA2 and lack of cross-reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression in six of seven B cell non-Hodgkin's lymphoma and one of six Burkitt's lymphoma cell lines. Reverse transcriptase-PCR experiments and Western blotting using MAb F25 confirmed the expression profile. We also found that HCL cells from five patients expressed IRTA2. CONCLUSIONS: Our results provide the first evidence that IRTA2 is expressed on the surface of human lymphoma cell lines and HCL cells. IRTA2 could be useful as a new target for immunotherapy.

Activating transcription factor 4 increases the cisplatin resistance of human cancer cell lines.

Resistance to cisplatin is a major problem in the treatment of solid tumors. To investigate the determinants of cisplatin resistance, we have identified cisplatin-inducible genes by differential display of mRNA. One of the cisplatin-inducible genes was identified as activating transcription factor 4 (ATF4). Northern blot analysis demonstrated that expression of ATF4 is inducible at the transcriptional level. Its expression is also up-regulated in two cisplatin-resistant cell lines. We tested whether cellular levels of ATF4 are responsible for cisplatin sensitivity by examining 11 human lung cancer cell lines. Expression of ATF4 was found to correlate with cisplatin sensitivity (P = 0.01). We also evaluated the cisplatin sensitivity of two stable transfectants overexpressing ATF4. Both were less sensitive to cisplatin than the parental cells but equally sensitive to vincristine. Our findings suggest that levels of ATF4 expression could help to predict cisplatin sensitivity.

Structural and functional characterization of two human V-ATPase subunit gene promoters.

The vacuolar-ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to proton pumping across membranes. V-ATPase genes are considered to be housekeeping genes and are expressed in human neoplastic tissue and in cell lines. We have isolated and characterized several genomic clones containing the 5'-end of the human V-ATPase genes. DNA sequence analysis of the promoters of two V-ATPase subunit genes, encoding C (ATP6C) and c (ATP6F), reveals GC-rich regions in the region of the first exon. Neither TATA- nor CCAAT-boxes were found in these promoters, but both GC-boxes and E-boxes were identified. Transient transfection analysis, using a series of 5' nested deletions of promoter-luciferase constructs in human cancer cells, demonstrated that a positive cis-acting regulatory region was present in these TATA-less promoters. The regions between -79 and -40 of the ATP6C promoter and between -245 and -99 of the ATP6F promoter were identified as being likely to be extremely important for basal promoter activity. Electrophoretic mobility shift assays (EMSA) of these cis-regulatory regions revealed the basal promoter to be highly complex, with cooperative binding of several transcription factors, including Sp family members. These data identify the critical regulatory regions for both the ATP6C and ATP6F basal promoters and stress the functional importance of multiple protein complexes, involving the Sp family of transcription factors, in regulating gene expression.

Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy.

One of the major obstacles to the successful treatment of cancer is the complex biology of solid tumour development. Although regulation of intracellular pH has been shown to be critically important for many cellular functions, pH regulation has not been fully investigated in the field of cancer. It has, however, been shown that cellular pH is crucial for biological functions such as cell proliferation, invasion and metastasis, drug resistance and apoptosis. Hypoxic conditions are often observed during the development of solid tumours and lead to intracellular and extracellular acidosis. Cellular acidosis has been shown to be a trigger in the early phase of apoptosis and leads to activation of endonucleases inducing DNA fragmentation. To avoid intracellular acidification under such conditions, pH regulators are thought to be up-regulated in tumour cells. Four major types of pH regulator have been identified: the proton pump, the sodium-proton exchanger family (NHE), the bicarbonate transporter family (BCT) and the monocarboxylate transporter family (MCT). Here, we describe the structure and function of pH regulators expressed in tumour tissue. Understanding pH regulation in tumour cells may provide new ways of inducing tumour-specific apoptosis, thus aiding cancer chemotherapy.

Rapid grouping of monoclonal antibodies based on their topographical epitopes by a label-free competitive immunoassay.

Topography of epitopes of monoclonal antibodies (MAbs), identified as the mutual competition of the MAbs, can be valuable indicators for the biological functions of MAbs. However, the determination of topographical epitopes is not performed before the functional screening of MAbs, because the requirement for purifying and labeling of MAbs makes the mapping experiment difficult, particularly in the early stage of MAb production. Here we describe a new label-free competitive enzyme-linked immunosorbent assay (LFC-ELISA) for the rapid grouping of MAbs based on the topography of their epitopes. In the LFC-ELISA, the immune complex formed by a competitor, MAb#2, and an antigen is challenged by an indicator, MAb#1 that had been captured on the ELISA plate through a secondary antibody. The MAb#2-antigen immune complex is trapped by MAb#1 only if MAb#1 reacts with an epitope different from that of MAb#2. The immune complex (MAb#2-antigen-MAb#1) is detected with an enzyme-labeled reagent specific to a tag on the antigen. Our experiments using different anti-CD30 MAbs and a CD30-Fc fusion protein as the antigen revealed that the LFC-ELISA performed well with MAbs of different isotypes (IgG1, IgG2a, and IgG2b), and in a practical range of MAb concentrations (0.3-10 microg/ml) and affinities (0.9-13 nM of Kd). We obtained pairwise competition data from all 26 anti-CD30 MAbs. We then utilized a cluster analysis and a bootstrap method to analyze the competition data for grouping of the MAbs. This objective and automated analysis identified eight distinct topographical epitopes on CD30. The reactivity of the anti-CD30 MAbs in immunoblot, and their inhibiting activity on CD30-CD30-ligand binding correlated with the topographical epitopes. The results show that the LFC-ELISA combined with cluster analysis is a useful new method for grouping MAbs based on their topographical epitopes and can be used in the early stage of MAb production. One useful application is to identify MAbs reacting with different epitopes from a large number of MAbs so that the most appropriate MAbs can be selected for therapeutic use.

CD21(-/low) marginal zone B cells highly express Fc receptor-like 5 protein and are killed by anti-Fc receptor-like 5 immunotoxins in hepatitis C virus-associated mixed cryoglobulinemia vasculitis.

OBJECTIVE: Hepatitis C virus (HCV) is associated with B cell lymphoproliferative disorders, including mixed cryoglobulinemia (MC) vasculitis and B cell non-Hodgkin's lymphoma. The expansion of clonal and autoreactive rheumatoid factor-bearing CD21(-/low) marginal zone (MZ) B cells was demonstrated in patients with HCV-associated MC vasculitis. Fc receptor-like (FCRL) proteins comprise a family of immunoregulatory proteins preferentially expressed on B lineage cells. The goal of this study was to investigate the expression of FCRL proteins 1-5 on B cells from patients with HCV-associated MC vasculitis. METHODS: Expression of FCRL proteins 1-5 was assessed by flow cytometry on B cells from 15 HCV-infected patients with type II MC (7 of whom had B cell non-Hodgkin's lymphoma), 20 HCV-infected patients without MC, and 20 healthy donors. To evaluate FCRL-5 as an immunotherapy target in HCV-associated MC vasculitis, 2 anti-FCRL-5 recombinant immunotoxins were produced using anti-FCRL-5 monoclonal antibodies and Pseudomonas exotoxin. RESULTS: Expression of FCRLs 2, 3, and 5 was markedly increased while expression of FCRL-1 was decreased on clonal CD21(-/low) MZ B cells, as compared with other B cell subsets, from HCV-infected patients and healthy donors. However, there was no difference in the pattern of FCRL expression between HCV-MC patients with lymphoma and those without lymphoma. The anti-FCRL-5 immunotoxins showed specific cytotoxicity against FCRL-5-expressing clonal CD21(-/low) MZ B cells isolated from HCV-infected patients as well as FCRL-5-transfected cell lines. No cytotoxicity against T cells or conventional B cells was observed. CONCLUSION: These findings suggest that FCRL-5-targeting therapies could be a specific treatment for HCV-associated MC vasculitis and other FCRL-5-positive autoimmune B cell disorders.

CD164 and FCRL3 are highly expressed on CD4+CD26- T cells in Sézary syndrome patients.

Sézary syndrome (SS) cells express cell surface molecules also found on normal activated CD4 T cells. In an effort to find a more specific surface marker for malignant SS cells, a microarray analysis of gene expression was performed. Results showed significantly increased levels of mRNA for CD164, a sialomucin found on human CD34+ hematopoietic stem cells, and FCRL3, a molecule present on a subset of human natural T regulatory cells. Both markers were increased in CD4 T cells from SS patients compared with healthy donors (HD). Flow cytometry studies confirmed the increased expression of CD164 and FCRL3 primarily on CD4+CD26- T cells of SS patients. Importantly, a statistically significant correlation was found between an elevated percentage of CD4+CD164+ T cells and an elevated percentage of CD4+CD26- T cells in all tested SS patients but not in patients with mycosis fungoides and atopic dermatitis or HD. FCRL3 expression was significantly increased only in patients with high tumor burden. CD4+CD164+ cells displayed cerebriform morphology and their loss correlated with clinical improvement in treated patients. Our results suggest that CD164 can serve as a marker for diagnosis and for monitoring progression of cutaneous T-cell lymphoma (CTCL)/SS and that FCRL3 expression correlates with a high circulating tumor burden.

Fc receptor-like 5 promotes B cell proliferation and drives the development of cells displaying switched isotypes.

The biological roles of B cell membrane proteins in the FCRL family are enigmatic. FCRL proteins, including FCRL5, were shown to modulate early BCR signaling, although the subsequent, functional consequences of receptor engagement are poorly understood. We found that FCRL5 surface protein itself was induced temporarily upon BCR stimulation of human, naive B cells, indicating precise control over timing of FCRL5 engagement. Cross-linking of FCRL5 on cells induced to express FCRL5 enhanced B cell proliferation significantly. This enhancement required costimulation of the BCR and TLR9, two signals required for optimal proliferation of naive B cells, whereas T cell help in the form of anti-CD40 and IL-2 was dispensable. In addition, we found that FCRL5 stimulation generated a high proportion of cells displaying surface IgG and IgA. Optimal development of cells expressing switched isotypes required T cell help, in addition to stimuli found necessary for enhanced proliferation. Surprisingly, cells that developed upon FCRL5 stimulation simultaneously displayed surface IgM, IgG, and IgA. Cells expressing multiple Ig isotypes were described in hairy cell leukemia, a disease in which FCRL5 is overexpressed. Enhanced proliferation and downstream isotype expression upon FCRL5 stimulation could reflect a physiological role for FCRL5 in the expansion and development of antigen-primed B cells. In addition, FCRL5 may promote growth of malignant cells in hairy cell leukemia and other FCRL5-expressing tumors.

FCRL1 on chronic lymphocytic leukemia, hairy cell leukemia, and B-cell non-Hodgkin lymphoma as a target of immunotoxins.

FCRL1 (Fc receptor-like 1) is a cell-surface membrane protein belonging to FCRL family and is preferentially expressed on B cells. To evaluate FcRL1 as an immunotherapy target for B-cell malignancies, we prepared anti-FCRL1 mAbs without cross-reactivity to other FCRL family proteins and analyzed FCRL1 protein expression on malignant cells from patients and on B-cell lines. Frequent FCRL1 expression was observed by flow cytometry on 12 B-cell non-Hodgkin lymphoma (B-NHL) cell lines and many patient samples: 12 of 14 chronic lymphocytic leukemia (CLL), 7 of 7 follicular lymphoma (FL), 13 of 17 hairy cell leukemia (HCL), and 2 of 3 mantle cell lymphoma (MCL). Two recombinant immunotoxins, E3(Fv)-PE38 and E9(Fv)-PE38, were constructed. Both immunotoxins bound to FCRL1-positive cells with similar affinities (3.4 and 3.2 nM) and were cytotoxic to cell lines, but E9(Fv)-PE38 was 4- to 20-fold more cytotoxic than E3(Fv)-PE38. The concentrations that inhibited response by 50% (IC(50)s) of E9(Fv)-PE38 on 11 different FCRL1-positive cell lines ranged from 1.0 ng/mL to 90 ng/mL and correlated with the FCRL1 expression levels. Our results suggest that anti-FCRL1 immunotoxin E9(Fv)-PE38 exhibits remarkably specific cytotoxicity and merits further evaluation for the treatment of FCRL1-positive malignancies, including CLL, HCL, FL, MCL, and other B-NHL.

Palmitoylation of POTE family proteins for plasma membrane targeting.

The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane.

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells.

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.