Increased expression of the RIalpha subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer.
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The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIalpha subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIalpha expression in patients with ovarian cancer. We have evaluated the expression of RIalpha in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIalpha mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIalpha mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIalpha mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIalpha mRNA expression and differentiation state. RIalpha mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIalpha mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIalpha mRNA expression is associated with advanced stage disease. RIalpha expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type. (+info)
Diminished levels of protein kinase A RI alpha and RI beta transcripts and proteins in systemic lupus erythematosus T lymphocytes.
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Deficient type I protein kinase A phosphotransferase activity occurs in the T cells of 80% of subjects with systemic lupus erythematosus (SLE). To investigate the mechanism of this deficient isozyme activity, we hypothesized that reduced amounts of type I regulatory (RI) isoform transcripts, RIalpha and RIbeta, may be associated with a diminution of RIalpha and/or RIbeta protein. Sixteen SLE subjects with a mean (+/-1 SD) SLE disease activity index of 12.4 +/- 7.2 were studied. Controls included 16 normal subjects, six subjects with primary Sjogren's syndrome (SS), and three subjects with SS/SLE overlap. RT-PCR revealed that normal, SS, SS/SLE, and SLE T cells expressed mRNAs for all seven R and catalytic (C) subunit isoforms. Quantification of mRNAs by competitive PCR revealed that the ratio of RIalpha mRNA to RIbeta mRNA in normal T cells was 3.4:1. In SLE T cells there were 20 and 49% decreases in RIalpha and RIbeta mRNAs (RIbeta; p = 0.008), respectively, resulting in an RIalpha:RIbeta mRNA of 5.3:1. SS/SLE T cells showed a 72.5% decrease in RIbeta mRNA compared with normal controls (p = 0.01). Immunoblotting of normal T cell RIalpha and RIbeta proteins revealed a ratio of RIalpha:RIbeta of 3.2:1. In SLE T cells, there was a 30% decrease in RIalpha protein (p = 0.002) and a 65% decrease in RIbeta protein (p < 0.001), shifting the ratio of RIalpha:RIbeta protein to 6.5:1. T cells from 25% of SLE subjects lacked any detectable RIbeta protein. Analysis of several lupus T cell lines demonstrated a persistent deficiency of both proteins, excluding a potential effect of disease activity. In conclusion, reduced expression of RIalpha and RIbeta transcripts is associated with a decrement in RIalpha and RIbeta proteins and may contribute to deficient type I protein kinase A isozyme activity in SLE T cells. (+info)
Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry: identification of Ser81 as the in vivo phosphorylation site of RIalpha.
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The mechanism by which the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RIalpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RIalpha gene product. A single RIalpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RIalpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RIalpha. Mass spectrometry also indicated that membrane-associated RIalpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RIalpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RIalpha strongly suggests an interaction between RIalpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RIalpha membrane association in the heart. (+info)
Protein kinase A-Ialpha subunit-directed antisense inhibition of ovarian cancer cell growth: crosstalk with tyrosine kinase signaling pathway.
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Expression of the RIalpha subunit of cAMP-dependent protein kinase type I is increased in human cancers in which an autocrine pathway for epidermal growth factor-related growth factors is activated. We have investigated the effect of sequence-specific inhibition of RIalpha gene expression on ovarian cancer cell growth. We report that RIalpha antisense treatment results in a reduction in RIalpha expression and protein kinase A type I, and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology and appearance of apoptotic nuclei. In addition, EGF receptor, c-erbB-2 and c-erbB-3 levels were reduced, and the basal and EGF-stimulated mitogen-activated protein kinase activities were reduced. Protein kinase A type I and EGF receptor levels were also reduced in cells overexpressing EGF receptor antisense cDNA. These results suggest that the antisense depletion of RIalpha leads to blockade of both the serine-threonine kinase and the tyrosine kinase signaling pathways resulting in arrest of ovarian cancer cell growth. (+info)
Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIalpha subunit of protein kinase A after oral administration.
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Overexpression of the RIalpha subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of (35)S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIalpha subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy. (+info)
A safety and pharmacokinetic study of a mixed-backbone oligonucleotide (GEM231) targeting the type I protein kinase A by two-hour infusions in patients with refractory solid tumors.
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GEM231 is a mixed-backbone oligonucleotide targeting the regulatory subunit alpha of type I protein kinase A, which plays an important role in growth and maintenance of malignancies. Preclinically, GEM231 inhibited human cancer xenografts either alone or synergistically with chemotherapeutic agents and has demonstrated an improved metabolic stability and safety profile compared to the first-generation compounds. Objectives of this study were to define the safety profile and pharmacokinetics of GEM231 administered as 2-h IV infusions twice weekly in patients with refractory solid tumors. Fourteen patients (13 evaluable for safety) received escalating doses of GEM231 at 20-360 mg/m2 (2.5-9 mg/kg). Tumor histologies included non-small cell lung cancer, renal cell cancer, sarcoma, and others. The plasma pharmacokinetics of GEM231 were linear and predictable. Maximum plasma concentration (Cmax) reached 50-70 microg/ml (8-13 microM) at dose 360 mg/m2 and 27-32 microg/ml at dose 240 mg/m2. The plasma half-life was about 1.5 h. The only clinical toxicities were transient grade I-II fever and fatigue at doses > or = 240 mg/m2. There was no treatment-related complement activation or thrombocytopenia at any dose level, except with the first dose in one patient who had pre-existing borderline thrombocytopenia. Transient activated partial thrombin time prolongation occurred at doses > or =160 mg/m2. Dose-limiting toxicities included transient activated partial thrombin time prolongation (one of three patients at 360 mg/m2) and cumulative reversible transaminase elevation (three of three patients at 360 mg/m2 and three of six patients at 240 mg/m2 during weeks 3-10). One patient with colon cancer had stabilization of a previously rising carcinoembryonic antigen. Thus, in this first clinical evaluation of a mixed-backbone oligonucleotide in cancer patients, high plasma concentrations of GEM231 were well tolerated without significant acute toxicities, but prolonged treatment was associated with reversible transaminitis. Although 240 mg/m2 by 2-h infusion twice weekly was safe for a 4-week treatment duration, alternative dosing schedules are being tested to minimize the cumulative toxicity, which will be essential to extend the duration of therapy at the highest GEM231 dose tested. (+info)
8-chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima formation induced by balloon injury in vivo.
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OBJECTIVES: The aims of the present study were to assess 1) the effect of 8-C1-cAMP (cyclic-3'-5'-adenosine monophosphate) on vascular smooth muscle cell (VSMC) proliferation in vitro and 2) the efficacy of systemic administration of 8-C1-cAMP on neointimal formation after balloon injury in vivo. BACKGROUND: Neointimal formation after vascular injury is responsible for restenosis after arterial stenting. Recently, 8-C1-cAMP, a cAMP analogue that induces growth arrest, has been safely administered in phase I studies in humans. METHODS: The effect of 8-C1-cAMP on cell proliferation was first assessed on SMCs in vitro. To study the effects of cAMP in vivo, balloon injury was performed in 67 rats using a 2F Fogarty balloon catheter. RESULTS: The 8-C1-cAMP markedly inhibited VSMC proliferation in vitro, reduced protein kinase A (PKA) RIalpha subunit expression, and induced PKA RIIbeta subunit expression. In addition, 8-C1-cAMP reduced, in a dose-dependent manner, neointimal area and neointima/media ratio after balloon injury. The proliferative activity, assessed by proliferating nuclear cell antigen immunostaining, revealed a reduction of proliferative activity of VSMCs in vivo in the 8-C1-cAMP group. Moreover, the systemic administration of 8-C1-cAMP did not affect renal function, blood pressure and heart rate. CONCLUSIONS: We conclude that 8-C1-cAMP potently inhibits VSMC proliferation in vitro and reduces neointima formation by balloon injury in vivo after systemic administration. These data may have a clinical relevance in designing future strategies to prevent restenosis after arterial stenting and perhaps after percutaneous transluminal coronary angioplasty. (+info)
Alternative 5'-exons of the mouse cAMP-dependent protein kinase subunit RIalpha gene are conserved and expressed in both a ubiquitous and tissue-restricted fashion.
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The activity of cAMP-dependent protein kinase is controlled by its regulatory subunits. Mouse RIalpha regulatory subunit expression is initiated from five different non-coding 5'-regions (exons 1a, 1b, 1c, 1d and 1e). This organization appears to be conserved among species. All mouse tissues accumulate exon 1a and 1b transcripts and most contain more 1b than 1a, except brain, heart and oesophagus. Exon 1d and 1e transcripts are found in several tissues, while exon 1c is testis-specific. All five transcripts are in RIalpha-rich tissues: gonads and adrenal glands. (+info)