Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RIalpha subunit

The primary mediator of cAMP action in mammalian cells is cAMP-dependent protein kinase (PKA). There are two types of PKA, type I (PKA-I) and type II (PKA-II), which share a common catalytic subunit but contain distinct regulatory subunits, RI and RII, respectively. Evidence suggests that increased expression of RIalpha/PKA-I correlates with neoplastic cell growth. Here, we show that sequence-specific oligonucleotide inhibition of RIalpha expression results in inhibition of growth and modulation of cAMP signaling in cancer cells. The antisense promoted growth inhibition in a time-dependent, concentration-dependent, and sequence-dependent manner in human cancer cells in monolayer culture, and it inhibited colony formation in soft agar and tumor growth in nude mice. Among the cancer cells are LS-174T, HCT-15, and Colo-205 colon carcinoma cells; A-549 lung carcinoma cells; LNCaP prostate adenocarcinoma cells; Molt-4 leukemia cells; and Jurkat T lymphoma cells. Northern blot and immunoprecipitation analyses revealed that the growth inhibitory effect of the antisense correlated with a decrease in RIalpha expression at both the mRNA and protein levels. Pulse-chase experiments revealed that the antisense-directed inhibition of RIalpha expression resulted in compensatory changes in expression of the isoforms of R and C subunits and cAMP signaling in a cell type-specific manner. These results demonstrate that cAMP is ubiquitous in the regulation of cell growth and that the antisense oligonucleotide, which inhibits the synthesis of the RIalpha subunit of PKA, can be targeted to a single gene for treatment of cancer in a variety of cell types.     

Cyclic Phosphatidic Acid Stimulates cAMP Production and Inhibits Growth in Human Colon Cancer Cells

Colon cancer is a malignancy that develops in colon and rectal tissues. The prognosis for metastatic colon cancer remains poor, and novel therapeutic options are required to reduce colon cancer mortality. Recently, intracellular cAMP levels have been suggested to influence the behavior of cancer cells. Intriguingly, cyclic phosphatidic acid (cPA) and its structural analogs inhibit growth in many cancer cell lines, and our previous work has suggested that cPA increases cAMP production. Phosphodiesterase (PDE) type 3 isoforms PDE3A and PDE3B are expressed mainly in cardiovascular tissue and adipose tissue, respectively. Moreover, increase in intracellular cAMP levels has been associated with the inhibition of growth in colon cancer cells. These findings suggest that cPA could be used in colon cancer therapy. In this study, we found that cPA inhibited the growth of HT-29 cells, which express high levels of PDE3B, but not the growth of DLD-1 cells, which express low levels of PDE3B. Furthermore, cPA inhibited the phosphorylation of Akt in HT-29 cells in a dose-dependent fashion. Our results suggest that PDE3B expression and intracellular cAMP levels are correlated with the proliferation of colon cancer cells. These findings demonstrate for the first time that cPA may serve as a useful a molecule in targeted therapy for colon cancer.     

Tumour Cells Expressing Single VEGF Isoforms Display Distinct Growth,Survival and Migration Characteristics

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.     

Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.     

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.     

Characterization of an orphan G protein-coupled receptor, GPR20, that constitutively activates Gi proteins

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E(2)-induced production of cAMP were significantly decreased. A remarkable increase in [(35)S]guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G(i/o) proteins. Involvement of GPR20 in the activation of G(i/o) proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [(35)S]GTPgammaS incorporation and inhibition of the prostaglandin E(2)-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [(3)H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G(i) proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling.     

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory G_i proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-x_L, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA⁻ mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA⁻ mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA⁻ mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.     

Differential effects of Cbl isoforms on Egfr signaling in Drosophila

The Cbl family of proteins downregulate epidermal growth factor receptor (Egfr) signaling via receptor internalization and destruction. These proteins contain two functional domains, a RING finger domain with E3 ligase activity, and a proline rich domain mediating the formation of protein complexes. The Drosophila cbl gene encodes two isoforms, D-CblS and D-CblL. While both contain a RING finger domain, the proline rich domain is absent from D-CblS. We demonstrate that expression of either isoform is sufficient to rescue both the lethality of a D-cbl null mutant and the adult phenotypes characteristic of Egfr hyperactivation, suggesting that both isoforms downregulate Egfr signaling. Interestingly, targeted overexpression of D-CblL, but not D-CblS, results in phenotypes characteristic of reduced Egfr signaling and suppresses the effect of constitutive Egfr activation. The level of D-CblL was significantly correlated with the phenotypic severity of reduced Egfr signaling, suggesting that D-CblL controls the efficiency of downregulation of Egfr signaling. Furthermore, reduced dynamin function suppresses the effects of D-CblL overexpression in follicle cells, suggesting that D-CblL promotes internalization of activated receptors. D-CblL is detected in a punctate cytoplasmic pattern, whereas D-CblS is mainly localized at the follicle cell cortex. Therefore, D-CblS and D-CblL may downregulate Egfr through distinct mechanisms.     

Natriuretic peptides increase cAMP production in human thyrocytes via the natriuretic peptide clearance receptor (NPR-C)

The relationship between natriuretic peptides and adenylyl cyclase/cAMP signal transduction has generally been shown to be an inhibitory one, mediated via the NPR-C receptor coupled to adenylyl cyclase by inhibitory G proteins (Gi). In the present studies, we have investigated the modulation of cAMP by natriuretic peptides in a long-term culture of human thyroid cells. Competition of [125I] rat ANF binding to human thyrocytes (HTU-5) by rat ANF (99-126) and by the NPR-C-specific analog C-ANF (4-23) indicated that greater than 97% of the ANF binding sites on HTU-5 cells are of the NPR-C type. However, rather than inhibiting intracellular cAMP in these cells, ANF increased maximal cAMP to 200-300% of control value. The ANF-induced increase in cAMP was duplicated by C-ANF (4-23). Basal cAMP content was reduced, and the response to ANF was abolished when the cells were grown in low (0.5%) serum without the addition of pituitary and hypothalamic extracts. CNP-22 also increased cAMP above control in HTU-5 cells identically to ANF. Neither ANF nor C-ANF (4-23) had any effect on cAMP in a culture of rat aortic smooth muscle cells. These results provide the first evidence for a positive effect of natriuretic peptides on cAMP mediated through the NPR-C, suggesting the possibility of an alternative mode of signaling by this receptor subtype.     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

Competitive cAMP antagonists for cAMP-receptor proteins

The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.     

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.     

Compartmentation of cAMP signaling in cardiac myocytes: a computational study

Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of beta(1)-adrenergic receptor (beta(1)AR) and M(2) muscarinic receptor (M(2)R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of beta(1)ARs and M(2)Rs, as well as the location of stimulatory (G(s)) and inhibitory (G(i)) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G(i), and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high ( approximately 1 microM) and only modestly stimulated by beta(1)AR activation ( approximately 2 microM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A ( approximately 100 nM to approximately 2 microM); 2), M(2)R activation strongly reduces the beta(1)AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak beta(1)AR stimulation, M(2)R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M(2)R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.