The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

Screening and identification of inhibitors of endoplasmic reticulum stress-induced activation of the IRE1α-XBP1 branch.

Endoplasmic reticulum (ER) stress and the subsequent adaptive cellular response, termed the unfolded protein response (UPR), have been implicated in several diseases, including cancer. In this review, I present a brief introduction to ER stress and the UPR and then summarize the importance of the IRE1α-XBP1 branch as a target for anticancer drug discovery. In addition, I introduce our approach to the identification of inhibitors against the IRE1α-XBP1 branch from microbial cultures. As a result of our screening, toyocamycin has been identified and toyocamycin showed anticancer activity against multiple myeloma.

Effect of povidone iodine, chlorhexidine digluconate and toyocamycin on amphizoic amoebic strains, infectious agents of Acanthamoeba keratitis - a growing threat to human health worldwide.

INTRODUCTION: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. MATERIAL AND METHODS: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. RESULTS: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. CONCLUSIONS: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.

Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice.

BACKGROUND AND AIMS: A high serum level of saturated free fatty acids (FFAs) is associated with the development of nonalcoholic fatty liver disease (NAFLD). X-box binding protein-1 (XBP-1) is activated by FFA treatment upon splicing. XBP-1 is a transcription factor induced by the endoplasmic reticulum (ER) stress sensor endoribonuclease inositol-requiring enzyme 1 alpha (IRE1α). However, the role of XBP-1 in NAFLD remains relatively unexplored. Toyocamycin was recently reported to attenuate the activation of XBP-1, possibly by inducing a conformational change in IRE1α. In this study, we examined the effect of toyocamycin on hepatocyte lipoapoptosis and steatosis. We also explored the effects of toyocamycin in a mouse model of NAFLD. METHODS: Huh-7 cells and isolated rat primary hepatocytes were treated with palmitic acid (PA), which is a saturated FFA, in the presence or absence of toyocamycin. In addition, male C57BL/6J mice were fed a diet rich in saturated fat, fructose, and cholesterol (FFC) for 4 months, after which the effect of toyocamycin was assessed. RESULTS: Toyocamycin attenuated FFA-induced steatosis. It also significantly reduced PA-induced hepatocyte lipoapoptosis. In addition, toyocamycin reduced the expression of cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein (CHOP), which is a key player in ER stress-mediated apoptosis, as well as its downstream cell death modulator, death receptor 5. In the in vivo study, toyocamycin ameliorated the liver injury caused by FFC-induced NAFLD. It also reduced hepatic steatosis and the expression of lipogenic genes. CONCLUSIONS: The data we obtained suggest that toyocamycin attenuates hepatocyte lipogenesis and ameliorates NAFLD in vivo and may therefore be beneficial in the treatment of NAFLD in humans.

Toyocamycin induces apoptosis via the crosstalk between reactive oxygen species and p38/ERK MAPKs signaling pathway in human prostate cancer PC-3 cells.

BACKGROUND: Toyocamycin, an antibiotic agent isolated from Streptomyces species, has been shown to have anticancer and chemopreventive effects on various cancer cells. Until now, Toyocamycin-induced apoptosis has not been reported to be involved in the regulation between mitogen-activated protein kinases (MAPKs) and reactive oxygen species (ROS) production. METHODS: Cell viability assay, western blot, cell-cycle arrest, annexin V/propidium iodide assay, reactive oxygen species (ROS) production, mitochondrial membrane potential and intracellular Ca2+ flux were assayed. RESULTS: We investigated the apoptotic effect of Toyocamycin and the underlying molecular mechanism in prostate cancer PC-3 cells. Toyocamycin treatment resulted in reduced cell viability of PC-3 cells, but not of non-malignant RWPE-1 cells. Toyocamycin enhanced apoptosis, mitochondrial dysfunction, and ROS production in PC-3 cells. In addition, MAPK proteins were activated upon Toyocamycin treatment. The p38 and extracellular signal-regulated kinases (ERK) activities were regulated by ROS-mediated signaling pathway underlying the Toyocamycin-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) recovered the Toyocamycin-induced mitochondrial dysfunction, ROS, and apoptosis. Additionally, p38 stimulated ROS production and inhibitory effects on ERK activation, while ERK inhibited the ROS production and had no effect on p38 activation. CONCLUSION: ROS-mediated activation of p38/ERK partially contributes to Toyocamycin-induced apoptosis, and p38/ERK MAPKs regulate the ROS production in PC-3 cells.

Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells.

Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.

Synthesis of purine and 7-deazapurine nucleoside analogues of 6-N-(4-Nitrobenzyl)adenosine; inhibition of nucleoside transport and proliferation of cancer cells.

Human equilibrative nucleoside transporter 1 (hENT1) is a prototypical nucleoside transporter protein ubiquitously expressed on the cell surface of almost all human tissue. Given the role of hENT1 in the transport of nucleoside drugs, an important class of therapeutics in the treatment of various cancers and viral infections, efforts have been made to better understand the mechanisms by which hENT1 modulates nucleoside transport. To that end, we report here the design and synthesis of novel tool compounds for the further study of hENT1. The 7-deazapurine nucleoside antibiotic tubercidin was converted into its 4-N-benzyl and 4-N-(4-nitrobenzyl) derivatives by alkylation at N3 followed by a Dimroth rearrangement to the 4-N-isomer or by fluoro-diazotization followed by SN Ar displacement of the 4-fluoro group by a benzylamine. The 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin antibiotics were prepared by the alkylation approach. Cross-membrane transport of labeled uridine by hENT1 was inhibited to a weaker extent by the 4-nitrobenzylated tubercidin and sangivamycin analogues than was observed with 6-N-(4-nitrobenzyl)adenosine. Type-specific inhibition of cancer cell proliferation was observed at micromolar concentrations with the 4-N-(4-nitrobenzyl) derivatives of sangivamycin and toyocamycin, and also with 4-N-benzyltubercidin. Treatment of 2',3',5'-O-acetyladenosine with aryl isocyanates gave the 6-ureido derivatives but none of them exhibited inhibitory activity against cancer cell proliferation or hENT1.

Natural compounds' activity against cancer stem-like or fast-cycling melanoma cells.

BACKGROUND: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. METHODS: We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. FINDINGS: Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. CONCLUSION: Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

Interaction of Rio1 kinase with toyocamycin reveals a conformational switch that controls oligomeric state and catalytic activity.

Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2'-deoxyadenosine.

This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.2 microM Mz while the clinically, highly Mz-resistant isolate has an MIC of 50-100 microM. This isolate was cross-resistant to other members of the 5-NI family of compounds including tinidazole and other experimental compounds and maintained resistance under anaerobic conditions. In addition, this isolate was cross-resistant to the 5-nitrothiazole compound nitazoxanide and the 5-nitrofuran derivative, furazolidone. Adenosine analogues toyocamycin and 2-fluoro-2'-deoxyadenosine with no nitro group were also less effective against the clinically Mz-resistant isolate than a Mz-susceptible one. Three other isolates which were determined to be Mz-resistant soon after isolation lost resistance in the long term. One other isolate has maintained some level of permanent Mz resistance (MIC of 25 microM). A multi-drug resistance mechanism may be involved in these clinically Mz-resistant isolates.

Cell cycle arrest and cytochrome c-mediated apoptotic induction in human lung cancer A549 cells by MCS-C2, an analogue of sangivamycin.

In the course of our screening for novel modulators on cell cycle progression and apoptosis as anticancer drug candidates, we generated an analogue of sangivamycin, MCS-C2, designated as 4-amino-6-bromo-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide. This study was aimed to evaluate the molecular mechanisms on cell cycle arrest and apoptotic induction of MCS-C2 in human lung cancer A549 cells. To investigate the effects of MCS-C2 on cell cycle progression in A549 cells, we measured DNA content of A549 cells treated with 5 microM of HY253 using flow cytometric analysis. The flow cytometric analysis revealed an appreciable G(2) phase arrest in A549 cells treated with 5 micronM of MCS-C2. This MCS-C2-induced G(2) phase arrest is associated with significant up-regulation of p53 and p21(Cip1) in A549 cells. Furthermore, TUNEL assay was used to examine apoptotic induction in A549 cells treated with 5 microM of MCS-C2 for 48 h. In addition, the effects of MCS-C2 on apoptosis-associated proteins in A549 cells were examined using Western blot analysis. The apoptotic induction in MCS-C2-treated A549 cells is associated with cytochrome c release from mitochondria which in turn resulted in the activation of caspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). In conclusion, based on these results, we suggest that MCS-C2 may be a potent cancer chemotherapeutic candidate for use in treating human lung cancer cells via up-regulation and activation of p53.

Hydrogenosomes of laboratory-induced metronidazole-resistant Trichomonas vaginalis lines are downsized while those from clinically metronidazole-resistant isolates are not.

Trichomonas vaginalis is the most common sexually transmitted protozoan in the world and its resistance to metronidazole is increasing. The purpose of this study was to demonstrate that clinical metronidazole resistance in T. vaginalis does not occur via the same mechanism as laboratory-induced metronidazole resistance--that is, via hydrogenosome down sizing. Ultrathin sections of this parasite were examined using transmission electron microscopy and the size and area of the cell and hydrogenosomes were compared between drug-resistant laboratory lines and clinically resistant isolates. Clinical metronidazole-resistant T. vaginalis had similar-sized hydrogenosomes as a metronidazole-sensitive isolate. Inducing metronidazole resistance in both of these isolates caused down sizing of hydrogenosomes. Inducing toyocamycin resistance did not cause any ultrastructural changes to the cell or to the hydrogenosome. No correlation between hydrogenosome number and the drug-resistant status of T. vaginalis isolates and lines was observed. This report demonstrates that clinical metronidazole resistance is not associated with down-sized hydrogenosomes, thus indicating that an alternative resistance mechanism is used by T. vaginalis.

Toyocamycin specifically inhibits auxin signaling mediated by SCFTIR1 pathway.

The auxins, plant hormones, play a crucial role in many aspects of plant development by regulating cell division, elongation and differentiation. Toyocamycin, a nucleoside-type antibiotic, was identified as auxin signaling inhibitor in a screen of microbial extracts for inhibition of the auxin-inducible reporter gene assay. Toyocamycin specifically inhibited auxin-responsive gene expression, but did not affect other hormone-inducible gene expression. Toyocamycin also blocked auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCF(TIR1) ubiquitin-proteasome pathway without inhibiting proteolytic activity of proteasome. Furthermore, toyocamycin inhibited auxin-induced lateral root formation and epinastic growth of cotyledon in the Arabidopsis thaliana plant. This evidence suggested that toyocamycin would act on the ubiquitination process regulated by SCF(TIR1) machineries. To address the structural requirements for the specific activity of toyocamycin on auxin signaling, the structure-activity relationships of nine toyocamycin-related compounds, including sangivamycin and tubercidin, were investigated.

Functional expression of ribozymes in Apicomplexa: towards exogenous control of gene expression by inducible RNA-cleavage.

The ability to control expression of a specific gene is a prerequisite to understand the function of essential genes. Many gene regulation systems operate on the transcriptional level by employing heterologous cis- and trans-acting elements. Recently, novel approaches employing autocatalytic RNA have been reported for different organisms. Here we show specific downregulation of gene expression in the apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum, employing self-cleaving ribozymes integrated into the transcriptional unit of different genes. Moreover, we demonstrate the potential to specifically upregulate reporter gene expression by employment of the recently identified ribozyme inhibitor toyocamycin. At the RNA-level, we were able to significantly stabilise the mRNA in T. gondii. Furthermore we show that the adenosine analogue toyocamycin needs to be phosphorylated by adenosine kinase in order to act as an inhibitor for hammerhead ribozymes, since neither upregulation of reporter gene expression nor a toxic effect of toyocamycin can be detected in parasites that do not express the enzyme adenosine kinase.

Synthesis of pyrrolo[2,3-d]pyrimidine nucleoside derivatives as potential anti-HCV agents.

Several Toyocamycin (4) analogues were examined for their ability to inhibit HCV RNA in a replicon assay. Among the compounds examined 4-methylthio (18) and 5-carboxamide oxime derivatives (23 and 27) of Toyocamycin were found to have good activity and selectivity.

Cell cycle arrest and apoptotic induction in LNCaP cells by MCS-C2, novel cyclin-dependent kinase inhibitor, through p53/p21WAF1/CIP1 pathway.

The purpose of the present study was to investigate the mechanisms involved in the antiproliferative and apoptotic effects of MCS-C2, a novel analog of the pyrrolo[2,3-d]pyrimidine nucleoside toyocamycin and sangivamycin, in human prostate cancer LNCaP cells. MCS-C2, a selective inhibitor of cyclin-dependent kinase, was found to inhibit cell growth in a time- and dose-dependent manner, and inhibit cell cycle progression by inducing the arrest of the G1 phase and apoptosis in LNCaP cells. When treated with 3 microM MCS-C2, inhibited proliferation associated with apoptotic induction was found in the LNCaP cells in a concentration and time-dependent manner, and nuclear DAPI staining revealed the typical nuclear features of apoptosis. Furthermore, MCS-C2 induced cell cycle arrest in the G1 phase through the upregulated phosphorylation of the p53 protein at Ser-15 and activation of its downstream target gene p21WAF1/CIP1. Accordingly, these results suggest that MCS-C2 inhibits the proliferation of LNCaP cells by way of G1-phase arrest and apoptosis in association with the regulation of multiple molecules in the cell cycle progression.

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.

Induction of apoptosis in human leukemia cells by MCS-C2 via caspase-dependent Bid cleavage and cytochrome c release.

The purpose of the present study was to investigate the anti-proliferative and apoptotic effects of MCS-C2, a novel synthetic analogue of the pyrrolo[2,3-d]pyrimidine nucleoside toyocamycin and sangivamycin, in human promyelocytic leukemia (HL-60) cells. When treated with 5 microM MCS-C2, inhibited proliferation associated with apoptotic induction was found in the HL-60 cells in a concentration-dependent and time-dependent manner, plus nuclear DAPI staining revealed the typical nuclear features of apoptosis. However, MCS-C2 showed almost no antiproliferative effect and no apoptotic induction in normal lymphocyte cells used as a control when compared with those in HL-60 cancer cells. Moreover, a flow cytometric analysis of the HL-60 cells using FITC-dUTP and propidium iodide (PI) showed that the apoptotic cell population increased gradually from <1% at 0 h to 34% at 12 h after exposure to 5 microM MCS-C2. This apoptotic induction was associated with the cleavage of Bid and a release of cytochrome c from mitochondria into the cytosol, followed by the activation of caspase-3 and inactivation of poly(ADP-ribose) polymerase (PARP). However, there was no significant change in any other mitochondrial membrane proteins, such as Bcl-2 and Bax. Consequently, the current findings suggest that the mitochondrial pathway was primarily involved in the MCS-C2-induced apoptosis in the human promyelocytic leukemia HL-60 cells.

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Induction of p16/INK4a gene expression and cellular senescence by toyocamycin.

We constructed an assay system of a luciferase reporter with p16/lNK4a gene transcriptional regulatory domain to identify p16-inducing substances, and found toyocamycin to induce gene expression from the screening of culture fluids of Streptomyces. Toyocamycin is a nucleoside analog, and it increased the p16 mRNA level in human normal fibroblasts or synovial cells as assessed by Northern blot hybridization or real time RT-PCR. It also induced cellular senescence in normal human fibroblasts. The transcriptional regulatory regions of human p16 gene that were responsible for the induction were analyzed using deletion mutants of the transcriptional regulatory region of p16 linked to the luciferase gene. The DNA fragment -111 to +1 bp from the cap site was sufficient to drive toyocamycin-activated transcription of p16/luciferase reporter. Nucleotide sequences within this domain contained the Sp1- and Ets-binding sequences. Mutations were introduced into these sequences, and the Sp1 sequence was found to be critical for the induction, and this notion was confirmed from gel-mobility shift assay.