ErbB-4 ribozymes abolish neuregulin-induced mitogenesis.
The epidermal growth factor-like receptor tyrosine kinase (ErbB) family is frequently overexpressed in a variety of human carcinomas, including breast cancer. To assist in characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes, Rz6, Rz21, and Rz29, efficiently catalyzed the specific cleavage of ErbB-4 message in a cell-free system. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells. Inhibition of mitogenesis was characterized by ribozyme-mediated down-regulation of ErbB-4 expression. In addition, we provide the first evidence that different threshold levels of ErbB-4 expression and activation correlate with different responses to neuregulin stimulation. High levels of ErbB-4 expression, phosphorylation, and homodimerization are necessary for neuregulin-stimulated, interleukin 3-independent cell proliferation in the 32D/E4 cells. In the case of Rz29-transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin-induced phosphorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 ribozyme in T47D human breast carcinoma cells led to a down-regulation of endogenous ErbB-4 expression and a reduction of anchorage-independent colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.
Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007-2197, USA. Tangc@gunet.georgetown.edu
C K
CK
Tang
D J
DJ
Goldstein
J
J
Payne
F
F
Czubayko
M
M
Alimandi
L M
LM
Wang
J H
JH
Pierce
M E
ME
Lippman
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002474:Cell-Free System; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D015536:Down-Regulation; D066246:ErbB Receptors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D006023:Glycoproteins / Q000494:pharmacology*; D006413:Hematopoietic System / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D020880:Neuregulins; D010766:Phosphorylation; D016337:RNA, Catalytic / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D066247:Receptor, ErbB-4; D013268:Stimulation, Chemical; D013379:Substrate Specificity
D006023:Glycoproteins; D007377:Interleukin-3; D020880:Neuregulins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D004247:DNA; C587689:ERBB4 protein, human; D066246:ErbB Receptors; C587690:Erbb4 protein, mouse; D066247:Receptor, ErbB-4