Characterization of anti-cyclin E single-chain Fv antibodies and intrabodies in breast cancer cells: enhanced intracellular stability of novel sFv-F(c) intrabodies.
Cyclin E is a critical cell cycle protein in the regulated progression of normal cells to replicate their DNA. Ectopic overexpression of cyclin E results in accelerated G(1) progression, chromosome instability, and a reduced requirement for growth factors. Dysregulated cyclin E expression is found in nearly all breast cancers examined. Toward the goal of developing a system to block cyclin E function in normal and breast cancer cells, we have developed anti-cyclin E single-chain antibodies (sFvs) for use as intrabodies. We have cloned the variable region genes from two hybridoma cell lines that produce anti-human cyclin E antibodies, linked them into sFvs, and showed their ability to bind cyclin E when expressed as sFv-F(c) fusion proteins. Engineering of the sFvs as sFv-F(c) intrabodies resulted in a dramatic increase in the sFv half-life as analyzed by pulse-chase and immunofluorescence, and these fusion intrabodies can be expressed in the cytosol or retargeted to the nucleus of breast cancer cell lines. Stable expression of a nuclear-targeted anti-cyclin E intrabody appears to inhibit the growth of the breast cancer cell line SKBR3. This work sets the stage for the development of intrabody-based inducible or tissue-specific cyclin E knockouts and for identifying cyclin E and its vital cell cycle functions as a potential gene therapy target in breast and other cancers.
Strube
Randall W
RW
Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27109, USA.
Chen
Si-Yi
SY
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology; D019556:COS Cells; D002455:Cell Division; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019927:Cyclin E / Q000276:immunology*; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007424:Intracellular Fluid / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D019927:Cyclin E; D018076:DNA, Complementary; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv