Characterization of anti-cyclin E single-chain Fv antibodies and intrabodies in breast cancer cells: enhanced intracellular stability of novel sFv-F(c) intrabodies.

摘要

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.