A novel antagonist to the inhibitors of apoptosis (IAPs) potentiates cell death in EGFR-overexpressing non-small-cell lung cancer cells.

In the effort to develop an efficient chemotherapy drug for the treatment of non-small-cell lung cancer (NSCLC), we analyzed the anti-tumorigenic effects of a novel small molecule targeting the inhibitor of apoptosis (IAPs), HM90822B, on NSCLC cells. HM90822B efficiently decreased IAP expression, especially that of XIAP and survivin, in several NSCLC cells. Interestingly, cells overexpressing epidermal growth factor receptor (EGFR) due to the mutations were more sensitive to HM90822B, undergoing cell cycle arrest and apoptosis when treated. In xenograft experiments, inoculated EGFR-overexpressing NSCLC cells showed tumor regression when treated with the inhibitor, demonstrating the chemotherapeutic potential of this agent. Mechanistically, decreased levels of EGFR, Akt and phospho-MAPKs were observed in inhibitor-treated PC-9 cells on phosphorylation array and western blotting analysis, indicating that the reagent inhibited cell growth by preventing critical cell survival signaling pathways. In addition, gene-specific knockdown studies against XIAP and/or EGFR further uncovered the involvement of Akt and MAPK pathways in HM90822B-mediated downregulation of NSCLC cell growth. Together, these results support that HM90822B is a promising candidate to be developed as lung tumor chemotherapeutics by targeting oncogenic activities of IAP together with inhibiting cell survival signaling pathways.

p34 is a novel regulator of the oncogenic behavior of NEDD4-1 and PTEN.

PTEN is one of the most frequently mutated or deleted tumor suppressors in human cancers. NEDD4-1 was recently identified as the E3 ubiquitin ligase for PTEN; however, a number of important questions remain regarding the role of ubiquitination in regulating PTEN function and the mechanisms by which PTEN ubiquitination is regulated. In the present study, we demonstrated that p34, which was identified as a binding partner of NEDD4-1, controls PTEN ubiquitination by regulating NEDD4-1 protein stability. p34 interacts with the WW1 domain of NEDD4-1, an interaction that enhances NEDD4-1 stability. Expression of p34 promotes PTEN poly-ubiquitination, leading to PTEN protein degradation, whereas p34 knockdown results in PTEN mono-ubiquitination. Notably, an inverse correlation between PTEN and p34/NEDD4-1 levels was confirmed in tumor samples from colon cancer patients. Thus, p34 acts as a key regulator of the oncogenic behavior of NEDD4-1 and PTEN.

Cyclophilin A is a secreted growth factor induced by oxidative stress.

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal-regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.

Stress pathway activation induces phosphorylation of retinoid X receptor.

Cellular stresses inhibit retinoid signaling, but the molecular basis for this phenomenon has not been revealed. Here, we present evidence that retinoid X receptor (RXR) is a substrate for both mitogen-activated protein kinase kinase-4 (MKK4/SEK1) and its downstream mediator c-Jun N-terminal kinase (JNK). MKK4/SEK1 and JNK recognized distinct features on RXR in the DE and AB regions, respectively. Phosphorylation by MKK4/SEK1 had profound effects on the biochemical properties of RXR, inhibiting the expression of genes activated by RXR-retinoic acid receptor complexes. Tyr-249 in the RXR DE region was required for the inhibitory effect of MKK4/SEK1. These effects were significantly reduced in MKK4/SEK1-null cells, indicating that MKK4/SEK1 is required for the suppression of retinoid signaling by stress. Findings presented here demonstrate that MKK4/SEK1 can directly modulate transcription by phosphorylating RXR, a novel MKK4/SEK1 substrate.

Cell transformation by the superoxide-generating oxidase Mox1.

Reactive oxygen species (ROS) generated in some non-phagocytic cells are implicated in mitogenic signalling and cancer. Many cancer cells show increased production of ROS, and normal cells exposed to hydrogen peroxide or superoxide show increased proliferation and express growth-related genes. ROS are generated in response to growth factors, and may affect cell growth, for example in vascular smooth-muscle cells. Increased ROS in Ras-transformed fibroblasts correlates with increased mitogenic rate. Here we describe the cloning of mox1, which encodes a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase of phagocytes, gp91phox. mox1 messenger RNA is expressed in colon, prostate, uterus and vascular smooth muscle, but not in peripheral blood leukocytes. In smooth-muscle cells, platelet-derived growth factor induces mox1 mRNA production, while antisense mox1 mRNA decreases superoxide generation and serum-stimulated growth. Overexpression of mox1 in NIH3T3 cells increases superoxide generation and cell growth. Cells expressing mox1 have a transformed appearance, show anchorage-independent growth and produce tumours in athymic mice. These data link ROS production by Mox1 to growth control in non-phagocytic cells.

Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera.

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).

Constructing an efficient trans-acting genomic HDV ribozyme.

We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP alpha S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25, 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

Identification of important bases in a single-stranded region (SSrC) of the hepatitis delta (delta) virus ribozyme.

Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (delta) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.

Systematic substitution of individual bases in two important single-stranded regions of the HDV ribozyme for evaluation of the role of specific bases.

To elucidate the role of specific bases in the self-cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single-stranded regions [between nucleotides 726-731 (SSrA region) and 762-766 (SSrB region)] were carried out by oligonucleotide-directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base-substitutions and self-cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found to be essential for the ribozyme activity. We could determine the preferred sequences, 5'-G-G-(G/A/U)-N-(A/U/G)-Pu-3' for SSrA and 5'-(G/U)-C-N-(A/G/U)-A-3' for SSrB regions, respectively.

Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.

Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme.

In elucidating the part played by the essential stem structures (I, II, and III) in the self-cleavage activity of genomic HDV ribozyme, several point and compensation variants were constructed on pseudoknot-like structure by site-directed mutagenesis. The self-cleavage activities of these variants indicated that stems I and III were essential for the activity by forming Watson-Crick base pairs. On the other hand, disruption of A704:U767 had little influence on the cleavage activity, indicating that it is not essential in forming an active structure. Also, our V1 nuclease probing studies showed that the A704U and HDV88 variants have a sensitivity similar to the nuclease, and major cuts are visible in the stem I and stem II regions. Thus, stem I and stem II regions are maintained together with stem III regions in both molecules. These results and our earlier site-directed mutagenesis studies strongly support a pseudoknot-like structure for the genomic HDV ribozyme.

Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme.

In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.

Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions.

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.