Establishment and some characteristics of epoxomicin (a proteasome inhibitor) resistant variants of the human squamous cell carcinoma cell line, A431.
A431 resistant variants to epoxomicin (EXM) were established, showing 4.0-6.7 times more resistance to EXM than parental A431P. Both variants demonstrated increased expression of the beta-subunit molecules of 26S proteasome with approximately 2.5 times increased activity. In variant cells, cyclin B and P34cdc2 were over-expressed, whereas P21WAF1 was expressed at a similar level to A431P. Because of the proteasome inhibitor acting as a G2/M blocker, results are to the advantage of resistant cells proliferating in the presence of an inhibitor under a severe environment. Variant cells showed increased expression of epidermal growth factor receptor (EGFR) and decreased expression of mRNA, but also slight accumulation of protein of c-Cbl, which is a negative regulator of EGFR possessing ubiquitin ligase activity to desensitize EGF signaling. UbcH7, acting intimately with c-Cbl, was decreased in level compared to A431P. These phenomena can be regarded as one of the causes of prevention of c-Cbl-mediated down-regulation of EGFR in variant cells, enabling them to live. The anti-apoptotic Bcl-2 mainly consisted of a phosphorylated form with resistance to proteasomal degradation, suggesting that Bcl-2 phosphorylation occurred independently of its apoptotic function. Variant cells showed resistance not only to EXM, but to the 5 proteasome inhibitors, while demonstrating collateral sensitivity to doxorubicin.
Ohkawa
Kiyoshi
K
Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan. pko@jikei.ac.jp
Asakura
Tadashi
T
Aoki
Katsuhiko
K
Shibata
Shun-Ichi
S
Minami
Jiro
J
Fujiwara
Chieko
C
Sai
Tomoaki
T
Marushima
Hideki
H
Kuzuu
Hirofusa
H
D017209:Apoptosis; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division; D045744:Cell Line, Tumor; D019926:Cyclin B / Q000378:metabolism; D003546:Cysteine Endopeptidases; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D016195:G2 Phase; D006801:Humans; D020128:Inhibitory Concentration 50; D008938:Mitosis; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009842:Oligopeptides / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D019926:Cyclin B; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D016203:CDC2 Protein Kinase; D010744:Phosphoric Monoester Hydrolases; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C067419:CBL protein, human; C078846:epoxomicin