Cyclin D1 is a key regulatory factor of the G1 to S transition during cell cycle progression. Aberrant
cyclin D gene amplification and abnormal
protein expression have been linked to
hepatocellular carcinoma (HCC)
tumorigenesis. Intrabodies, effective anticancer
therapies that specifically inhibit target
protein function within all intracellular compartments, may block
cyclin D1 function. Here, a
single-chain variable fragment (scFv) antibody against
cyclin D1 (ADκ) selected from a human semi-synthetic phage display scFv library is expressed in Escherichia coli as soluble ADκ. Purified ADκ specifically binds to recombinant and endogenous
cyclin D1 with high affinity. To enable blocking of intracellular
cyclin D1 activity, an endoplasmic reticulum (ER) retention
signal sequence is added to the ADκ sequence to encode anti-
cyclin D1 intrabody ER-ADκ. Transfection of HepG2 cells with expression vector encoding ER-ADκ elicited intracellular ER-ADκ expression leading to
cyclin D1 binding, significant G1 phase arrest, and apoptosis that are mechanistically tied to decreased intracellular phosphorylated
retinoblastoma protein (Rb) levels. Meanwhile, ER-ADκ dramatically inhibited subcutaneous human HCC xenografts growth in nude mice in vivo after injection of
tumors with expression vector encoding ER-ADκ. These results demonstrate the potential of intrabody-based
cyclin D1 targeting
therapy as a promising treatment for HCC.