MiR-107 and MiR-185 Can Induce Cell Cycle Arrest in Human Non Small Cell Lung Cancer Cell Lines

BACKGROUND: MicroRNAs (miRNAs) are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5' seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. CONCLUSIONS/SIGNIFICANCE: We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term 'cell cycle'. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors

Apoptotic Volume Decrease (AVD) Is Independent of Mitochondrial Dysfunction and Initiator Caspase Activation

Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation

Roles of Disordered Volume Regulation in Apoptosis

A major hallmark of apoptosis is normotonic shrinkage of cells. Here, we studied the relation between apoptotic cell shrinkage and apoptotic cell death. Induction of the apoptotic volume decrease (AVD) under normotonic conditions was found to be coupled to facilitation of the regulatory volume decrease (RVD), which is known to be attained by parallel operation of Cl- and K+ channels, under hypotonic conditions. Both the AVD induction and the RVD facilitation were found to precede cytochrome c release, initiator caspase (-8, -9) activation, effector caspase (-3) activation, DNA laddering and ultrastructural alterations in four cell types (human lymphoid U937, epithelial HeLa, rat pheochromocytoma PC12 and mouse neuroblastoma X rat glioma hybrid NG108-15 cells) after apoptotic insults with two distinct apoptosis inducers. Also, the AVD was not prevented by a broad-spectrum caspase inhibitor. When the AVD induction and the RVD facilitation were prevented by blocking volume-regulatory Cl- or K+ channels, these cells did not show succeeding apoptotic biochemical and morphological events and were rescued from death. Thus, it is concluded that the AVD, which is caused by disordered cell volume regulation, is an early prerequisite to apoptotic events leading to cell death. It was previously reported that hypertonic stress triggers apoptosis in cell types that lack the regulatory volume increase (RVI). In fact, in Na+/H+ antiporter -deficient CCL39 cells, which were found to lack the RVI mechanism, hypertonic stimulation was found to induce persistent cell shrinkage without following volume recovery, and then lead to succeeding apoptotic events and cell death. When the RVI ability was conferred by transfection of the gene of NHE1 isoform of Na+/H+ antiporter, this cell line failed to exhibit apoptosis under hypertonic stimulation. Taken together, it is concluded that disordered or altered cell volume regulation is a prerequisite to apoptosis

Swelling-activated, cystic fibrosis transmembrane conductance regulator-augmented ATP release and Cl− conductances in murine C127 cells

A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin-luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling-induced ATP release by 10–20 times under hypotonic conditions (≤ 80 % osmolality).Glibenclamide failed to suppress swelling-induced ATP release from C127/CFTR cells. In contrast, whole-cell patch-clamp recordings showed that both the cAMP-activated ohmic Cl− currents and volume-sensitive outwardly rectifying (VSOR) Cl− currents were prominently suppressed by glibenclamide.Gd3+ markedly blocked swelling-induced ATP release but failed to suppress both cAMP- and swelling-activated Cl− currents in the CFTR-expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl− currents were activated normally.The continuous presence of an ATP-hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl− currents in C127/CFTR cells.The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells.On balance, the following conclusions can be deduced. First, swelling-induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl− channel. Second, swelling-induced ATP release is not mediated by the VSOR Cl− channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl− channels in C127/CFTR cells