The
POTE family genes encode a highly homologous group of primate-specific proteins that contain ankyrin repeats and coiled coil domains. At least 13 paralogous
POTE family genes are found on 8 human ...chromosomes (2, 8, 13, 14, 15, 18, 21 and 22), which can be sorted into 3 groups based on sequence similarity. We identified by a database search a group of additional human ankyrin repeat domain proteins, of which ANKRD26 and ANKRD30A are the best characterized; these are more distant homologs of POTE family proteins. A comprehensive comparison of the genomic organization indicates that
ANKRD26 has the genomic structure of the possible ancestor of
ANKRD30A and all
POTE family genes. Extensive remodeling involving segmental loss and internal duplication appears to have reshaped the
ANKRD30A and
POTE family genes after the primal duplication of the ancestor gene. We also identified a mouse homolog of human ANKRD26, but failed to find a mouse homolog that bears the structural characteristics of any of the POTE family of proteins. The mouse Ankrd26 may serve as a useful model for the study of the function of human ANKRD26, ANKRD30A and POTE family proteins.
Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially ...purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl β-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine > daunomycin > actinomycin D > verapamil > colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 μ M) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 μ mol per min per mg of protein).
The incidence of neoplastic meningitis is on the rise. Neoplastic meningitis can result from a direct seeding of the neuraxis by primary brain tumors or by hematogeneous spread of systemic solid ...tumors. A frequent genetic alteration in primary brain tumors such as gliomas is an in-frame deletion in the epidermal growth factor receptor (EGFR) gene EGFRvIII, which brings together what were normally distant polypeptide sequences in the intact receptor. A novel glycine is formed at the fusion junction, resulting in a unique and tumor-specific target. By using phage display, we have isolated a single-chain antibody specific for the EGFRvIII mutation and expressed it with a modified form of the Pseudomonas exotoxin to form the immunotoxin MR1scFvPE38KDEL (MR-1). The multiple dose toxicity and therapeutic efficacy of MR-1 immunotoxin were tested in an athymic rat model of neoplastic meningitis. The maximally tolerated doses in non-tumor-bearing rats were three doses of 3 microg each. For therapeutic studies, the target was a neoplastic meningitis induced by intrathecal inoculation of the EGFRvIII-expressing human glioma U87MG.deltaEGFR. A dose escalation study compared the survival of three equal doses of 1, 2, and 3 microg of MR-1 immunotoxin with saline or 3 microg of the control immunotoxin specific for the interleukin 2 receptor, anti-Tac. All animals treated with three doses of saline or 3 microg of anti-Tac died, with median survival of 7 and 10 days, respectively. There were 75% (six of eight) long-term survivors in the group treated with three doses of 1 microg and 57% (four of seven) long-term survivors in the groups treated with three doses of either 2 or 3 microg of MR-1 immunotoxin. None of the MR-1 immunotoxin-treated groups reached median survival by the termination of the study at 53 days. Therefore, median survival was estimated to be >53 days, resulting in an estimated increase in median survival of >657% compared with saline and 430% versus anti-Tac. Compartmental therapy with three doses of 2 microg of MR-1 immunotoxin is effective in the treatment of EGFRvIII-expressing neoplastic meningitis. This dose was found to have no clinical or histopathological effects on non-tumor-bearing animals. MR-1 immunotoxin is, therefore, considered specific and safe within its therapeutic window. Phase I clinical trials for tumors invading the intrathecal space that express the EGFRvIII target should be initiated.
BACKGROUND: Glioblastoma (GBM) remains uniformly lethal despite the progress of conventional therapies, which lack specificity and thus result in high toxicity to normal cells. Immunotargeting ...therapy such as recombinant immunotoxin (RIT) is an alternative and a Phase I clinical trial of MR1-1-PE38KDEL is undergoing at Duke University Medical Center. To avoid the production of neutralizing antibodies against toxin portion and to allow for repeat treatment cycles, a less immunogenic form of PE, LR-LO10, has been generated by silencing the human B cell epitopes on PE38 and removing the immunodominant T cell epitopes. We have previously developed an affinity-matured glycoprotein NMB (GPNMB)-specific RIT, F6V-PE38, which exhibits significant in vitro and anti-tumor activity against GBM and melanomas. Here we report a newly engineered F6V-LR-LO10 RIT for efficient, targeted therapy of GBM and other GPNMB-expressing cancer patients with normal immunity. METHODS: We have constructed an RIT, F6V-LR-LO10, by fusing a fully human F6V scFv, as VH-(G4S)3-VL, with a truncated form of Pseudomonas exotoxin A, LR-LO10. F6V-LR-LO10 was expressed in E. coli BL21 (DE3), and the inclusion bodies were reduced, refolded, and purified by ion exchange chromatography and gel filtration. The antigen binding of F6V-LR-LO10 was assessed by surface plasmon resonance and flow cytometric analysis. The cytotoxicity was measured by protein inhibition assay on GPNMB-expressing GBM and melanoma cells. The in vivo animal toxicity was tested in nude mice and anti-tumor activity will be assessed in human xenograft mouse models. RESULTS: The F6V-LR-LO10 protein was purified as a monomer of 51 kDa to 95% homogeneity. The F6V-LR-LO10 bound to GPNMB-coated chips with high affinity at KD = 1.1e-8 M. The F6V-LR-LO10 also bound to native GPNMB protein via flow cytometric analysis on GPNMB positive cell lines. We tested the in vitro activity of F6V-LR-LO10 on various glioma and melanoma xenograft cells and demonstrated cell-killing activity. The IC50 of F6V-LR-LO10 on GBM D392 cells was 1.5 ng/mL; the IC50 was 10 ng/mL and 15 ng/mL on melanoma DM443 and DM440 cells, respectively, and that was comparable to the IC50 of F6V-PE38, while negative control RIT did not kill those cells. The animal toxicity (MTD) was greatly reduced by >17 fold for F6V-LR-LO10 than F6V-PE38. CONCLUSIONS: Our experiments showed that a new fully human anti-GPNMB RIT, F6V-LR-LO10, is highly active with higher therapeutic index and is predicted to have low immunogenicity in humans. This next-generation RIT is suitable for further clinical development in therapy of GBM, melanoma, and other GPNMB-expressing malignancies. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.
Copines are ubiquitously expressed, phospholipid-binding proteins that have been conserved through evolution. In this paper, we report the cloning and molecular characterization of a new member of ...the Copine family, Copine 8. This gene has been isolated and characterized using a combination of bioinformatic and experimental approaches. Using an algorithm to cluster ESTs (expressed sequence tags) that are available through the public “GoldenPath” database, Copine 8 was initially identified as a gene predominantly expressed in prostate and testis. Cloning and molecular analysis revealed that this gene is expressed in low-levels in most tissues examined. Two different isoforms of this gene have been isolated. Strongest expression of Copine 8 mRNA is seen in the prostate, heart, and brain. Taken together, this data suggest that Copine 8 may have an important role to play in prostate regulation and development.
Chromosomal rearrangements resulting in gene fusions are frequently involved in carcinogenesis. Here, we describe a semiautomatic procedure for identifying fusion gene transcripts by using publicly ...available mRNA and EST databases. With this procedure, we have identified 96 transcript sequences that are derived from 60 known fusion genes. Also, 47 or more additional sequences appear to be derived from 20 or more previously unknown putative fusion genes. We have experimentally verified the presence of a previously unknown IRA1/RGS17 fusion in the breast cancer cell line MCF7. The fusion gene encodes the full-length RGS17 protein, a regulator of G protein-coupled signaling, under the control of the IRA1 gene promoter. This study demonstrates that databases of ESTs can be used to discover fusion genes resulting from structural rearrangement of chromosomes.
We have combined computer-based screening and experimental expression analysis to identify genes that are expressed in normal prostate and/or prostate cancer but not in essential human tissues. Using ...this approach we identified a new gene that is specifically expressed in testis, prostate, and placenta. The gene has one major transcript of 1.0
kb in size and encodes for a protein of 30.7
kDa molecular weight. We named this gene
TEPP (expressed in testis, prostate, and placenta). The amino acid sequence analysis of TEPP using SignalP program shows that it has a signal peptide with a predicted cleavage site between amino acids 19 and 20, indicating that it might be a secreted protein. Analysis of the predicted TEPP orthologs from different species shows that these proteins are highly conserved in chordates. In addition we have identified a splice variant of
TEPP, which encodes a 37
kDa protein. In conclusion, a combination of bioinformatic and molecular approaches is useful in the identification of genes expressed in specific tissues. Selective expression of
TEPP in testis, prostate, and in placenta and its high conservation among different species indicate that
TEPP might have a role in reproductive biology.
Highly active antiretroviral therapy (HAART), which combines multiple inhibitors of essential human immunodeficiency virus type 1 (HIV-1) enzymes, induces dramatic and sustained viral load reductions ...in many people infected with HIV-1. However, reservoirs of infected cells capable of producing replication-competent virus persist even after years of HAART, preventing elimination of infection. CD4-PE40 and 3B3(Fv)-PE38, chimeric toxins designed to target the HIV envelope (Env), represent a complementary class of agents that selectively kill productively infected cells. To investigate whether these Env-targeted toxins might serve as adjuncts to HAART for the elimination of infected cells, we tested their ability to augment HAART efficacy in vivo by using a thy/liv SCID-hu mouse model. CD4-PE40 and 3B3(Fv)-PE38 markedly enhanced the capacity of HAART to suppress acute HIV-1 infection and improved HAART-mediated viral load reduction in mice with established HIV-1 infection. These results represent the first demonstration of in vivo anti-HIV-1 efficacy for Env-targeted toxins and support their potential therapeutic utility in combination with HAART.
About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant ...immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the K
D
of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10
−9
M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC
50
was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.
Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic ...agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured 3Hvinblastine (3HVBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more 3HVBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of 3Hdexamethasone, to which the cells are equally sensitive, was observed. The difference in 3HVBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of 3HVBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit 3HVBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.
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