Expression cloning of cDNAs that render cancer cells resistant to Pseudomonas and diphtheria toxin and immunotoxins.
BACKGROUND
Several immunotoxins in which antibodies are coupled to plant or bacterial toxins are now in clinical trials for the treatment of cancer. One of these is B3-LysPE38 in which MAb B3 which reacts with many human cancers, is coupled with a genetically modified form of Pseudomonas exotoxin (PE).


METHODS
To investigate how cells can become resistant to PE-derived immunotoxins, we constructed an immunotoxin-sensitive MCF-7 breast cancer cell line that contains SV40 T antigen and allows episomal replication of SV40 origin containing plasmids. We transfected a pCDM8/HeLa cDNA expression library into these cells, thereby causing over-expression of the plasmid-encoded genes. The transfected cells were treated with immunotoxin to select for resistance-mediating plasmids, which were reisolated from these cells and amplified in Escherichia coli. The resulting plasmid pool was transfected into cells for two further rounds of selection and plasmid reisolation.


RESULTS
Several plasmids that caused immunotoxin resistance were enriched by this selection procedure. Four plasmids were stably transfected into MCF-7 cells and found to increase their resistance to PE-derived immunotoxins by 5- to 20-fold. These plasmids also confer resistance to native PE and to diphtheria toxin but not to ricin or cycloheximide. Thus, they appear to specifically interfere with the action of ADP-ribosylating toxins.


CONCLUSIONS
Cancer cells can become resistant to immunotoxins by deregulated expression of normal genes. The clinical significance of this type of resistance will be evaluated in clinical trials.
Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis, and Centers, Bethesda, Maryland, USA.
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Brinkmann
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Brinkmann
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Pastan
D036002:ADP Ribose Transferases; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis; D001427:Bacterial Toxins; D001943:Breast Neoplasms; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004261:DNA Replication; D018076:DNA, Complementary; D004167:Diphtheria Toxin / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005098:Exotoxins / Q000633:toxicity*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D010957:Plasmids; D011550:Pseudomonas aeruginosa; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000952:Antigens, Polyomavirus Transforming; D001427:Bacterial Toxins; D018076:DNA, Complementary; D004167:Diphtheria Toxin; D005098:Exotoxins; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A