Yun Gyu Park

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide GENE-SPECIFIC INHIBITION OF TUMOR GROWTH

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CRE- and AP-1-directed transcriptionin vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.

Glycogen synthase kinase 3β phosphorylates p21WAF1/CIP1 for proteasomal degradation after UV irradiation

ABSTRACT UV irradiation has been reported to induce p21WAF1/CIP1 protein degradation through a ubiquitin-proteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3β (GSK-3β) is required for its degradation in response to UV irradiation and that GSK-3β activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3β activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3β. ser-114, located within the putative GSK-3β target sequence, was phosphorylated by GSK-3β upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3β is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3β as the missing link between UV-induced ATR activation and p21 degradation.

Identification of potential lung cancer biomarkers using an in vitro carcinogenesis model

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799) all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.

Dissecting the Circuitry of Protein Kinase A and cAMP Signaling in Cancer Genesis

Abstract: Expression of the RIα subunit of the cAMP‐dependent protein kinase type I (PKA‐I) is enhanced in human cancer cell lines, in primary tumors, in transformed cells, and in cells upon stimulation of growth. Signaling via the cAMP pathway may be complex, and the biological effects of the pathway in normal cells may depend upon the physiological state of the cells. However, results of different experimental approaches such as antisense exposure, 8‐Cl‐cAMP treatment, and gene overexpression have shown that the inhibition of RIα/PKA‐I exerts antitumor activity in a wide variety of tumor‐derived cell lines examined in vitro and in vivo. cDNA microarrays have further shown that in a sequence‐specific manner, RIα antisense induces alterations in the gene expression profile of cancer cells and tumors. The cluster of genes that define the “proliferation‐transformation” signature are down‐regulated, and those that define the “differentiation‐reverse transformation” signature are up‐regulated in antisense‐treated cancer cells and tumors, but not in host livers, exhibiting the molecular portrait of the reverted (flat) phenotype of tumor cells. These results reveal a remarkable cellular regulation, elicited by the antisense RIα, superimposed on the regulation arising from the Watson‐Crick base‐pairing mechanism of action. Importantly, the blockade of both the PKA and PKC signaling pathways achieved with the CRE‐transcription factor decoy inhibits tumor cell growth without harming normal cell growth. Thus, a complex circuitry of cAMP signaling comprises cAMP growth regulatory function, and deregulation of the effector molecule by this circuitry may underlie cancer genesis and tumor progression.

Antisense depletion of RIα subunit of protein kinase A induces apoptosis and growth arrest in human breast cancer cells

In recent years, several laboratories have explored the possibility of using antisense oligodeoxynucleotides for specific manipulation of gene expression leading to cancer treatment. The enhanced expression of the RIα subunit of cyclic AMP-dependent protein kinase type I (PKA-I) has been correlated with cancer cell growth. In the present study, the effects of an antisense oligodeoxynucleotide targeted against RIα subunit of PKA-I on growth inhibition and apoptosis in MDA-MB-231 human breast cancer cells were investigated. The growth inhibitory effects of RIα antisense oligodeoxynucleotide correlated with a decrease in the RIα mRNA and protein levels. The growth inhibition was accompanied by changes in the cell cycle phase distribution, cell morpbology, cleavage of poly (ADP-ribose) polymerase (PARP), and appearance of apoptotic nuclei. By comparison, mismatched control oligodeoxynucleotide had no effect. On the basis of these results, it can be suggested that the RIα antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RIα and induces apoptosis/differentiation, could be used as a therapeutic agent for breast cancer treatment.

Genome-wide expression profiling of 8-chloroadenosine- and 8-chloro-cAMP-treated human neuroblastoma cells using radioactive human cDNA microarray

Previous reports raised question as to whether 8-chloro-cyclic adenosine 3,5-monophosphate (8-Cl-cAMP) is a prodrug for its metabolite, 8-Cl-adenosine which exerts growth inhibition in a broad spectrum of cancer cells. The present study was carried out to clarify overall cellular affects of 8-Cl-cAMP and 8-Cl-adenosine on SK-N-DZ human neuroblastoma cells by ystematically characterizing gene expression using radioactive human cDNA microarray. Microarray was prepared with PCR-amplified cDNA of 2,304 known genes spotted on nylon membranes, employing (33)P-labeled cDNAs of SK-N-DZ cells as a probe. the expression levels of approximately 100 cDNAs, representing about 8% of the total DNA elements on the array, were altered in 8-Cl-adenosine- or 8-Cl-cAMP-treated cells, respectively. The genome-wide expression of the two samples exhibited partial overlaps; different sets of up-regulated genes but the same set of down-regulated genes. 8-Cl-adenosine treatment up- egulated genes involved in differentiation and development (LIM protein, connexin 26, neogenin, neurofilament triplet L protein and p21(WAF1/CIP1)) and immune response such as natural killer cells protein 4, and down-regulated ones involved in proliferation and transformation (transforming growth factor-β, DYRK2, urokinase-type plasminogen activator and proteins involved in transcription and translation) which were in close parallel with those by 8-Cl-cAMP. Our results indicated that the two drugs shared common genomic pathways for the down-regulation of certain genes, but used distinct pathways for the up-regulation of different gene clusters. Based on the findings, we suggest that the anti-cancer activity of 8-Cl-cAMP results at least in part through 8-Cl-adenosine. Thus, the systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in anticancer activities of chemotherapeutics.

A genomic-scale view of the cAMP response element-enhancer decoy: A tumor target-based genetic tool

Enhancer DNA decoy oligodeoxynucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. Here, we use DNA microarrays to analyze the global effects of the CRE-decoy ODN in cancer cell lines and in tumors grown in nude mice. The CRE-decoy up-regulates the AP-2β transcription factor gene in tumors but not in the livers of host animals. The up-regulated expression of AP-2β is clustered with the up-regulation of other genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation is down-regulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. The CRE-decoy ODN, therefore, may serve as a target-based genetic tool to treat cancer and other diseases in which CRE-directed transcription is abnormally used.

Requirement of the cyclic adenosine monophosphate response element‐binding protein for hepatitis B virus replication

The cyclic adenosine monophosphate–response element (CRE)‐transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver‐specific transcription of viral genes and contains a CRE sequence (HBV‐CRE); however, whether the HBV‐CRE and CRE‐binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence‐comparison analysis of 984 HBVs reported worldwide showed that the HBV‐CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV‐CRE site was markedly inhibited by oligonucleotides containing HBV‐CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three‐base mutation into the HBV‐CRE led to a marked reduction in HBV messenger RNA synthesis. Conclusion: Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV‐CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection. (HEPATOLOGY 2008.)

CRE-decoy oligonucleotide-inhibition of gene expression and tumor growth

Nucleic acid molecules with high affinities for a target transcription factor can be introduced into cells as decoy cis-elements to bind these factors and alter gene expression. This review discusses a synthetic single-stranded palindromic oligonucleotide, which self-hybridizes to form a duplex/hairpin and competes with cAMP response element (CRE) enhancers for binding transcription factors. This oligonucleotide inhibits CRE- and Ap-1-directed gene transcription and promotes growth inhibition in vitro and in vivo in a broad spectrum of cancer cells, without adversely affecting normal cell growth. Evidence presented here suggests that the CRE-decoy oligonucleotide can provide a powerful new means of combating cancers, viral diseases, and other pathological conditions by regulating the expression of cAMP-responsive genes.

Interleukin-4 induces senescence in human renal carcinoma cell lines through STAT6 and p38 MAPK

Background: IL-4 can directly inhibit growth of several tumor cell types, but the molecular mechanism is not known. Results: IL-4 induces senescence by increasing p21WAF1/CIP1 expression through STAT6 and p38 MAPK. Conclusion: STAT6 and p38 MAPK play important roles in senescence induction by IL-4. Significance: This is the first report of cellular senescence induction by IL-4 and the responsible mechanism. Interleukin (IL)-4, originally identified as a lymphocyte growth factor, can directly inhibit growth of certain tumor cell types. We reported previously that IL-4 induced cell cycle arrest in G1 phase through an increase in p21WAF1/CIP1 expression in human renal cell carcinoma (RCC) cell lines. In the present study, we investigated the underlying mechanism of IL-4-induced growth inhibition. In four of six human RCC cell lines, including Caki-1, A498, SNU482, and SNU228, IL-4 induced cellular senescence as demonstrated by enlarged and flattened morphology, increased granularity, and senescence-associated-β-galactosidase (SA-β-gal) staining. Signal tranducer and activator of transcription 6 (STAT6) and p38 MAPK were found to mediate IL-4-induced growth inhibition and cellular senescence. Both of these molecules were activated by 10 min after IL-4 treatment, and inhibition of their activity or expression prevented growth suppression and cellular senescence induced by IL-4. Inhibiting or silencing either STAT6 or p38 MAPK alone partially reduced the effect of IL-4, whereas inhibiting or silencing both molecules exerted an additive effect and almost completely abrogated the effect of IL-4. Thus STAT6 and p38 MAPK appeared to independently mediate IL-4-induced growth inhibition and cellular senescence. The p21WAF1/CIP1 up-regulation that accompanied growth inhibition and cellular senescence by IL-4 was also attenuated additively when p38 MAPK and STAT6 were silenced. Taken together, these results show that IL-4 induces cellular senescence through independent signaling pathways involving STAT6 and p38 MAPK in some human RCC cell lines.