Mechanisms of the growth-inhibitory effect of the RNase-EGF fused protein against EGFR-overexpressing cells.
BACKGROUND
We previously showed the usefulness of a fused protein of human pancreatic ribonuclease1 (hRNase1) with human epidermal growth factor (hEGF) for molecular targeting of EGF receptor (EGFR)-overexpressing cells. In this study, the mechanisms underlying the inhibition of cell growth by RNase-EGF fused proteins was confirmed.


METHODS
Des.1-7 hRNase1 was genetically fused to hEGF. The fused proteins were expressed and isolated from Escherichia coli. The internalization of hRNase1-hEGF was confirmed by confocal fluorescence microscopy. The growth-inhibitory effect of the fused proteins was evaluated by MTT assay.


RESULTS
FITC-labelled hRNase1-hEGF was internalized into EGFR-overexpressing A431 cells. The internalization was not observed in A431 cells pre-treated with hEGF and EGFR-deficient H69 cells. The growth-inhibitory effect of des.1-7 hRNase1-hEGF against A431 cells was statistically significantly more pronounced than that of hRNase1-hEGF.


CONCLUSIONS
RNase-EGF fused proteins are internalized through EGFR and inhibit cell growth by exerting their ribonucleolytic activity in the cytosol.
Hoshimoto
Sojun
S
Department of Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan.
Ueda
Masakazu
M
Jinno
Hiromitsu
H
Kitajima
Masaki
M
Futami
Junichiro
J
Seno
Masaharu
M
D000595:Amino Acid Sequence; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D014846:Vulvar Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology
D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D012259:Ribonuclease, Pancreatic; D016650:Fluorescein-5-isothiocyanate