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 CREand AP-1-directed transcription in 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.Eukaryotic transcription is regulated by the interplay of various protein factors at promoters (1, 2). It has been shown that prokaryotic repressors can function as negative regulators of eukaryotic promoters (3,4). This observation suggests that displacement of activating proteins might provide a general strategy for gene-specific repression in eukaryotes. Several approaches have been undertaken to control eukaryotic gene expression through such displacement.In one approach, trans-dominant mutants are generated that interfere with the function of transactivators. Mutants are generated that retain the ability to bind to cis-regulatory DNA sequences but that have dysfunctional transcriptional activation domains. These mutant transcription factors compete with their functional, wild-type counterparts for binding to the enhancer sequences and prevent the activation or repression of the target gene. Although this strategy has been successful, in vitro (5-8), the generation of such mutants is not always possible. The transcription factor must be well characterized such that the activation domain(s) is identified and can be mutated. Also, even with sufficient knowledge to generate such mutants, difficult gene therapy procedures would be required to express these proteins in vivo.Promoter competition strategy has also been utilized whereby plasmids containing cis-acting elements in common with the targeted gene are introduced in high copy number into cells (9). At high copy number, a majority of the transcription factors can be competitively bound away from the native enhancer sequences with gene expression accordingly regulated. Because these plasmids must be stably maintained at high copy number in target cells, a requirement that is difficult to achieve in vivo, this approach has also been limiting.Another alternative is to employ oligonucleotides to form triple heli...
Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the C␣ or RI␣ subunit gene of PKA in an expression vector, which upregulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RII subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the C␣ gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH 2-terminal myristyl group of C␣ is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.
UV irradiation has been reported to induce p21WAF1/CIP1 protein degradation through a ubiquitinproteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3 (GSK-3) is required for its degradation in response to UV irradiation and that GSK-3 activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3 activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3. ser-114, located within the putative GSK-3 target sequence, was phosphorylated by GSK-3 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-3 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-3 as the missing link between UV-induced ATR activation and p21 degradation.
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.
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