Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.
ADP Ribose Transferases ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cell Proliferation ; drug effects ; Exotoxins ; genetics ; pharmacology ; Granzymes ; genetics ; pharmacology ; HeLa Cells ; Humans ; Recombinant Fusion Proteins ; pharmacology ; Virulence Factors ; genetics ; pharmacology

OBJECTIVETo study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the human bladder cancer T24 cells.

METHODSExpression of epidermal growth factor receptor (EGFR) in cultured T24 cells was analyzed with Western blot assay. Human bladder cancer T24 cells were exposed to TP40 at 5 - 1 000 microg/L. Methyl thiazolyl tetrazolium assay was applied to evaluate the cell proliferation by measuring the absorbance (A) at 570 nm with a microplate reader. Tritium labeled thymine deoxyriboside ([(3)H]-TdR) uptake was measured to observe DNA synthesis. Competition assays were performed by the EGF at 1 - 7 500 microg/L.

RESULTSExpression of EGFR was high in human bladder cancer T24 cells. Cell growth was suppressed by 10%, 19%, 27%, 41%, 47%, 53% and 61% after 96 h treatment with TP40 at 5, 50, 100, 250, 500, 750 and 1 000 microg/L, respectively. [(3)H]-TdR incorporation was 80%, 69%, 48% and 51% after 24 h, 48 h, 72 h, 96 h treatment with TP40 at 750 microg/L, respectively. When the concentration was 1 - 7 500 microg/L, EGF could block the inhibitory effect of TP40 to some extent.

CONCLUSIONSHuman bladder cancer T24 cells express EGFR at a high level. TP40 could inhibit the growth of T24 cells effectively in a dose- and time-dependent manner. The cytotoxic effects of TP40 were specifically mediated by EGFR.

Cell Proliferation ; drug effects ; Exotoxins ; pharmacology ; Humans ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Fusion Proteins ; pharmacology ; Transforming Growth Factor alpha ; pharmacology ; Tumor Cells, Cultured ; Urinary Bladder Neoplasms ; metabolism ; pathology

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology

ADP Ribose Transferases ; chemistry ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Bacterial Toxins ; chemistry ; pharmacology ; Exotoxins ; chemistry ; pharmacology ; Female ; HMGN2 Protein ; chemistry ; pharmacology ; HeLa Cells ; Humans ; Immunotoxins ; pharmacology ; Mice ; Mice, Inbred BALB C ; Protein Structure, Tertiary ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; Virulence Factors ; chemistry ; pharmacology

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.
Adult ; Bacteremia/*microbiology ; Bacterial Proteins/genetics ; Bacterial Toxins/*blood ; Exotoxins/*blood ; Female ; Humans ; Korea ; Leukocidins/*blood ; Male ; Methicillin/pharmacology ; Methicillin Resistance/drug effects ; Middle Aged ; Polymerase Chain Reaction/methods ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.
Adult ; Bacteremia/*microbiology ; Bacterial Proteins/genetics ; Bacterial Toxins/*blood ; Exotoxins/*blood ; Female ; Humans ; Korea ; Leukocidins/*blood ; Male ; Methicillin/pharmacology ; Methicillin Resistance/drug effects ; Middle Aged ; Polymerase Chain Reaction/methods ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.
Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; metabolism ; Humans ; Interleukin-15 ; antagonists & inhibitors ; biosynthesis ; genetics ; K562 Cells ; Pseudomonas aeruginosa ; genetics ; metabolism ; Receptors, Interleukin-15 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR).

METHODSA recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C.

RESULTSTo BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function.

CONCLUSIONThe protocol for targeting gene therapy against cancer with EGFR has been established successfully.

ADP Ribose Transferases ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Base Sequence ; Cell Line, Tumor ; Cells ; DNA ; genetics ; Exotoxins ; genetics ; pharmacology ; Gene Targeting ; Genetic Therapy ; Genetic Vectors ; Histones ; genetics ; Humans ; Molecular Sequence Data ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Transfection ; Virulence Factors ; genetics ; pharmacology

The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.
ADP Ribose Transferases ; chemistry ; metabolism ; pharmacology ; Amino Acid Sequence ; Blotting, Western ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Exotoxins ; chemistry ; metabolism ; pharmacology ; Humans ; Interleukin-6 ; chemistry ; metabolism ; pharmacology ; Molecular Sequence Data ; Pseudomonas aeruginosa ; genetics ; metabolism ; Recombinant Fusion Proteins ; chemistry ; metabolism ; pharmacology ; Sequence Analysis, Protein ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.
ADP Ribose Transferases/*pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Bacterial Toxins/*pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell/drug therapy/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Chromatography, Liquid ; Cyclin B/metabolism ; Cyclin-Dependent Kinase 2/metabolism ; Drug Resistance, Neoplasm/*drug effects ; E2F1 Transcription Factor/metabolism ; Electrophoresis ; Exotoxins/*pharmacology ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Mouth Neoplasms/drug therapy/*metabolism ; Tandem Mass Spectrometry ; Tumor Suppressor Protein p53/metabolism ; Virulence Factors/*pharmacology