ABSTRACT
Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.
Subject(s)
Cell Division/drug effects , Enzyme Inhibitors/pharmacology , Telomerase/antagonists & inhibitors , Gene Expression Profiling , Humans , Neoplasms/genetics , Neoplasms/pathology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Telomere , Tumor Cells, CulturedABSTRACT
Pin1, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases) has been implicated in the G2-M transition of the mammalian cell cycle. Pin1 interacts with a series of mitotic phosphoproteins, including Polo-like kinase-1, Cdc25C, and Cdc27, and is thought to act as a phosphorylation-dependent PPIase for these target molecules. Pin1 recognizes phosphorylated serine-proline or threonine-proline peptide-bonds in test substrates up to 1300-fold better than in the respective unphosphorylated peptides. To test directly whether Pin1 regulates the G2-M transition and/or progression through mitosis by catalyzing phosphorylation-dependent prolyl isomerization of essential mitotic targets, we examined the consequences of Pin1 depletion, achieved by (a) overexpression of Pin1 antisense RNA, (b) overexpression of dominant-negative Pin1, and (c) by a known small-molecule Pin1-PPIase inhibitor, juglone. The results of all of the three lines of investigation show that the catalytic activity of Pin1 is essential for tumor cell survival and entry into mitosis.
Subject(s)
Cell Survival , Mitosis , Peptidylprolyl Isomerase/metabolism , Proline/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis , Blotting, Western , Catalysis , Cell Line , Cell Nucleus/metabolism , Chromatin/metabolism , Cloning, Molecular , Enzyme Inhibitors/pharmacology , Histones/metabolism , Humans , Interphase , Kinetics , Microscopy, Fluorescence , Mutation , NIMA-Interacting Peptidylprolyl Isomerase , Naphthoquinones/pharmacology , Paclitaxel/pharmacology , Peptidylprolyl Isomerase/antagonists & inhibitors , Peptidylprolyl Isomerase/chemistry , Phosphorylation , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
The growth of solid neoplasms requires the recruitment of a supporting stroma. In most epithelial cancers, this stromal compartment comprises newly formed blood vessels and abundant, reactive stromal fibroblasts. Tumor stromal fibroblasts are not transformed but differ from resting fibrocytes in normal adult tissues by an altered pattern of gene expression. In human cancers, this includes induction of the cell-surface-bound fibroblast-activation protein (FAP), a member of the serine protease family encoded by the FAP gene on chromosome 2. In this study, we have cloned a complementary DNA for Fap, the murine homologue of FAP. The predicted murine FAP protein, mFAP, shares 89% amino-acid-sequence identity with human FAP, including a perfectly conserved catalytic triad. Cultured mouse embryo fibroblasts and mouse embryonic tissues were found to express Fap transcripts. In addition, the host-derived, fibroblast-rich stroma of human epithelial-cancer xenografts grown in immunodeficient mice also expresses Fap. Sequencing of reverse-transcription-PCR products indicates that 3 distinct Fap splice variants can be detected in tissues. Our findings suggest a close similarity in structure and tissue expression of FAP in different species. By extending the analysis of FAP to the mouse, new in vivo test systems become available for genetic and therapeutic manipulations and for the study of FAP regulation and function in embryonic development and in epithelial cancers.
