Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation sites were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting synthetic fragments encoding two and six phosphorylation sites into an expression vector, pdHL7. The phosphorylation sites were created by using the predicted consensus sequences for phosphorylation by the cAMP-dependent protein kinase to the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified antibodies (MAb-chCC49K1 and MAb-chCC49-6P) were expressed in NS0 cells and purified. The MAb-chCC49K1 protein contains two phosphorylation sites per heavy chain whereas the MAb-chCC49-6P protein contains six sites per heavy chain. Both MAb-chCC49K1 and MAb-chCC49-6P proteins can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity. The 32P-labeled MAb-chCC49K1 and MAb-chCC49-6P proteins bind to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into a monoclonal antibody provides a reagent for the diagnosis and treatment of cancer. The use of multiple phosphorylation sites provides antibodies with very high specific radioactivity and demonstrates that cassettes of phosphorylation sites can be introduced into proteins without altering their functional activity.
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PMID:Construction of phosphorylatable chimeric monoclonal antibody CC49. 962 12

A phosphorylation site for a tyrosine kinase was introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (Tyr) encoding one tyrosine kinase phosphorylation site into an expression vector. The phosphorylation site was created by incorporating the predicted consensus sequences for phosphorylation by the tyrosine kinase at the carboxyl terminus of the heavy chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49Tyr) was expressed and purified. The MAb-chCC49Tyr protein can be phosphorylated by the tyrosine Src kinase with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49Tyr protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into monoclonal antibodies (MAb) provides a new reagent for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, a tyrosine phosphorylation site can also be used to introduce phosphorylation sites into proteins.
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PMID:Construction of phosphorylatable chimeric monoclonal antibody CC49 with a tyrosine srC kinase recognition site. 973 2

8-Chloro-cyclic AMP (8-Cl-cAMP), a site-selective cAMP analogue, is a specific inhibitor of type I cAMP-dependent protein kinase (PKAI) and induces growth inhibition in several human and rodent tumor cell lines. The anti-epidermal growth factor receptor (EGFR) mAb 528 is a blocking antibody able to inhibit the in vitro and in vivo growth of several human cancer cell lines that express functional EGFRs. Since enhanced levels of PKAI are generally found in tumor cells and an increase in PKAI expression is induced by transformation through a transforming growth factor alpha/EGFR autocrine pathway, we have evaluated whether treatment with mAb 528 in combination with 8-Cl-cAMP may have an additive or synergistic growth inhibitory effect on human cancer cells. A dose-dependent inhibition of monolayer cell growth was observed in two human colon cancer cell lines (GEO and CBS) and in a human breast cancer cell line (MDA-468) by treatment with either mAb 528 or 8-Cl-cAMP with 50% inhibitory concentration of 2-10 microgram/ml or 20-25 micrometer, respectively. The combined treatment with low noninhibitory doses of mAb 528 (0.25 microgram/ml) and with 8-Cl-cAMP had a more than additive growth inhibitory effect with a 3- to 5-fold reduction in the 8-Cl-cAMP 50% inhibitory concentration in all cell lines tested. This combined treatment was similarly effective in inhibiting the soft agar cloning efficiency of GEO cells. 8-Cl-cAMP treatment of GEO cells induced a dose-dependent increase in cell membrane-associated EGFRs with a maximum 3- to 4-fold increase within 48-72 h of treatment. These results suggest that a double blockade of the PKAI serine-threonine kinase-dependent and of the EGFR tyrosine kinase-dependent pathways is potentially useful in cancer therapy.
Clin Cancer Res 1995 Feb
PMID:Cooperative antiproliferative effects of 8-chloro-cyclic AMP and 528 anti-epidermal growth factor receptor monoclonal antibody on human cancer cells. 981 69

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), a site-selective cyclic adenosine 3',5'-monophosphate (cAMP) analogue, exhibits growth inhibition in a broad spectrum of cancer cell lines. We investigated the effect of 8-Cl-cAMP on c-ras-transformed mouse fibroblasts (MP3/3T3) which were established by transfection of Balb3T3 cells (Balb3T3) with the point-mutated c-ras gene [G12-->V12]. 8-Cl-cAMP (2-5 microM) exerted over 80% growth inhibition by day 4 on MP3/3T3, while inhibiting parental Balb3T3 cell growth less than 40%. In order to distinguish the effect of 8-Cl-cAMP from that of 8-chloroadenosine (8-Cl-adenosine), we examined the effect of 8-Cl-cAMP in serum-free medium. 8-Cl-cAMP demonstrated a potent growth inhibition of MP3/3T3 cells cultured in serum-free medium, suggesting that the growth inhibitory effect of 8-Cl-cAMP was not due to its hydrolysed product, 8-Cl-adenosine. In addition, both Balb3T3 and MP3/3T3 contained cAMP phosphodiesterases mainly composed of isozyme IV which has previously been reported to be insensitive towards the hydrolysis of 8-Cl-cAMP. Non-transformed Balb3T3 cells contained only type II cAMP-dependent protein kinase (PKA), whereas transformed MP3/3T3 exhibited a marked increase in type I PKA. The growth inhibition of MP3/3T3 by 8-Cl-cAMP accompanied almost complete elimination of type I PKA without affecting type II PKA. Moreover, 8-Cl-cAMP induced an arrest in the G0/G1-phase of the cell cycle in MP3/3T3. 8-Cl-adenosine had little or no effect on the cell cycle kinetics of MP3/3T3 cells. These results show that 8-Cl-cAMP is a novel cAMP analogue which selectively eliminates type I PKA to induce growth inhibition in transformed fibroblasts.
Eur J Cancer 1998 Jul
PMID:8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) selectively eliminates protein kinase A type I to induce growth inhibition in c-ras-transformed fibroblasts. 984 89

The emergence of cisplatin resistance poses a significant problem to the treatment of a variety of human malignancies. Therefore, understanding the molecular basis of cisplatin resistance could improve the clinical effectiveness of this anticancer agent. Recently, our laboratory has demonstrated that cAMP-dependent protein kinase (PKA) mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells exhibited increased resistance to cisplatin as well as other DNA-damaging drugs. Further studies showed that either the functional inactivation of PKA or the mutation in the regulatory subunit gene may cause increased recognition of cisplatin-damaged DNA and enhanced DNA repair capacity. In this study, we evaluated the role of PKA in modulating cellular sensitivity to cisplatin in a series of PKA mutants of Saccharomyces cerevisiae. Mutants with decreased kinase activity resulting from a srv2 mutation showed no alterations in cisplatin sensitivity. Complementation of TPK1 in a yeast strain containing mutant tpk1 and also tpk2 and tpk3 deletions did not significantly alter its sensitivity to this DNA-damaging agent. Yeast transformants containing increased kinase activity resulting from overexpression of RAS2Val19 or TPK1 and yeast strains having increased kinase activities due to mutations in the BCY1 gene also did not show alterations in their sensitivity to cisplatin. Therefore, results from these studies unambiguously demonstrate that changes in PKA activity have no effect on cisplatin sensitivity in Saccharomyces cerevisiae.
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PMID:Cisplatin sensitivity in cAMP-dependent protein kinase mutants of Saccharomyces cerevisiae. 985 82

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.
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PMID:Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site. 1002 74

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.
Br J Cancer 1999 Feb
PMID:Increased expression of the RIalpha subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer. 1007 Aug 93

8-Cl-cAMP, a cAMP analogue that antagonizes type I cAMP-dependent protein kinase, is a novel anti-tumor agent presently under investigation in clinical trials. Herein we report the effects of this agent on epidermal growth factor receptor expression and degradation in human KB cancer cells. Exposure to 10 microM 8-Cl-cAMP for 48 h induced a 65% increase in epidermal growth factor receptor surface expression while the receptor synthesis was 22-fold enhanced. Analysis of epidermal growth factor-dependent receptor internalization in 8-Cl-cAMP-treated cells showed a higher endocytosis rate as well as an accelerated degradation which occurred together with an increased receptor ubiquitination. The enhanced degradation of epidermal growth factor receptor correlated with the lack of epidermal growth factor-induced proliferation and mitogen-activated protein kinase stimulation. The disregulation of epidermal growth factor receptor internalization and ubiquitin-dependent degradation could underlay a new mechanism of the anti-tumor activity of 8-Cl-cAMP suggesting its combination with agents that disrupt epidermal growth factor receptor signalling.
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PMID:Up-regulated EGF receptors undergo to rapid internalization and ubiquitin-dependent degradation in human cancer cells exposed to 8-Cl-cAMP. 1021 46

The cyclic AMP (cAMP)-dependent protein kinase regulatory subunit RI is overexpressed in cancer cells. 8-Chloro-cAMP (8-Cl-cAMP) is an RII site-specific analogue that down-regulates RI and inhibits the growth of a wide range of cancer cells in vitro and in vivo. We performed a Phase I trial of 8-Cl-cAMP in 32 patients with malignancies that were refractory to standard treatments. 8-Cl-cAMP was initially given in a 1-month cycle by constant infusion at 0.005 mg/kg/h for 21 days, followed by 1 week of rest. The dose was escalated to 0.045 mg/kg/h, but hypercalcemia became the dose-limiting toxicity. The length of drug administration was, therefore, reduced to 5 days per week for the first 3 weeks of the cycle, but it was not possible to increase the drug dose without producing hypercalcemia. Hence, the length of drug administration was reduced to 3 days per week for the first 3 weeks of the cycle. The maximum tolerated dose for this regimen was 0.15 mg/kg/h, and the dose-limiting toxicities were reversible hypercalcemia and hepatotoxicity. Stable disease for > or =4 months was observed in two patients treated at > or =0.045 mg/kg. cAMP-dependent protein kinase is involved in hormone- and cytokine-mediated signaling, and so representative hormone, cytokine, and peripheral lymphocyte subsets were measured. The drug had a parathyroid hormone-like effect on calcium homeostasis and significantly increased circulating luteinizing hormone and 17-hydoxyprogesterone levels (P < 0.02 and P < 0.0006, respectively). We conclude that 8-Cl-cAMP is well tolerated without attendant myelotoxicity, and in this study, it was associated with biological effects. In Phase II studies, a dose of 0.11 mg/kg/h for 3 days per week would be appropriate.
Clin Cancer Res 1999 Jul
PMID:Phase I study of the novel cyclic AMP (cAMP) analogue 8-chloro-cAMP in patients with cancer: toxicity, hormonal, and immunological effects. 1043 69

The drug transporter P-glycoprotein (P-gp) appears to play an important role in the ability of tumor cells to evade killing by chemotherapeutic agents. Using pharmacological inhibitors of cAMP-dependent protein kinase (PKA), it has been suggested that, similar to rodent model systems, the human P-gp gene (MDR1) is also under PKA-dependent control and that PKA inhibition may prove useful in reducing drug resistance in human cancer cells. To test this hypothesis, we stably transformed doxorubicin (Adriamycin)-resistant human MCF-7 breast cancer cells (MCF-7(ADR)) with a vector that inhibits PKA activity by inducing over-expression of mutant type Ialpha PKA regulatory (RIalpha) subunits. Two transformants (MCF-7(ADR-A) and MCF-7(ADR-B)) were found to express mutant RIalpha subunits and to possess markedly reduced PKA activity; another transformant (MCF-7(ADR-9)) lacked mutant RIalpha subunit expression and exhibited no inhibition of PKA activity. In contrast with findings in Chinese hamster ovary and Y1 adrenal cells, P-gp levels and cellular sensitivity to drugs which are P-gp substrates were unchanged in the PKA-inhibited transformants, suggesting that P-gp expression and function are not under PKA-dependent control in MCF-7(ADR) cells. Growth and saturation densities of the cell lines were highly correlated with level of PKA catalytic activity, suggesting that PKA inhibition may prove useful in inhibiting growth of breast tumor cells, even upon establishment of resistance to doxorubicin. However, our results challenge current proposals that drug sensitivity in P-gp-expressing human tumor cells may be restored by blocking MDR1 gene expression through inhibition of PKA activity.
Int J Cancer 1999 Sep 09
PMID:Lack of modulation of MDR1 gene expression by dominant inhibition of cAMP-dependent protein kinase in doxorubicin-resistant MCF-7 breast cancer cells. 1044 59


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