ABSTRACT
The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails. Furthermore, a significant proportion of C2H2-ZNF mRNAs are retained in the nucleus. Intriguingly, both short poly(A) tails and nuclear retention can be specified by the repeated elements that encode zinc finger motifs. These Zinc finger Coding Regions (ZCRs) appear to restrict polyadenylation of nascent RNAs and at the same time impede their export. However, the polyadenylation process is not necessary for nuclear retention of ZNF mRNAs. We propose that inefficient polyadenylation and export may allow C2H2-ZNF mRNAs to moonlight as non-coding RNAs or to be stored for later use.
Subject(s)
Active Transport, Cell Nucleus , CYS2-HIS2 Zinc Fingers , Cell Nucleus/metabolism , Polyadenylation , RNA Transport , RNA, Messenger/metabolism , Repetitive Sequences, Nucleic Acid , Cell Nucleus/genetics , Humans , RNA, Messenger/geneticsABSTRACT
BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines. METHODS: The effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules. RESULTS: AR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt. CONCLUSIONS: These results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical evaluation of HDAC inhibitors. HDAC inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in both canine and human OS.
Subject(s)
Bone Neoplasms/metabolism , Caspases/metabolism , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Osteosarcoma/metabolism , Phenylbutyrates/pharmacology , Animals , Bone Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dogs , Doxorubicin/pharmacology , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Osteosarcoma/drug therapy , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , VorinostatABSTRACT
Modifications of mRNAs can have a profound effect on cellular function and differentiation. In this issue of Cell Stem Cell, Batista et al. (2014) describe fundamental parameters of N(6)-methyl-adenosine modification of mRNAs in embryonic stem cells and provide strong evidence that modification plays a role in exit from pluripotency toward differentiation.
Subject(s)
Adenine/analogs & derivatives , Embryonic Stem Cells/physiology , Homeodomain Proteins/metabolism , Methyltransferases/metabolism , Animals , Female , HumansABSTRACT
While the phenomenon of polyadenylation has been well-studied, the dynamics of poly(A) tail size and its impact on transcript function and cell biology are less well-appreciated. The goal of this review is to encourage readers to view the poly(A) tail as a dynamic, changeable aspect of a transcript rather than a simple static entity that marks the 3' end of an mRNA. This could open up new angles of regulation in the post-transcriptional control of gene expression throughout development, differentiation and cancer.
