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)

The effects of steroids and peptide growth factors on aromatase activity in an androgen sensitive human prostate cancer cell line (LNCaP) were investigated. Factors were selected based on their observed modulation of the enzyme in other tissues. Incubation with epidermal growth factor and transforming growth factor-I decreased aromatase activity in LNCaP cells by 25-40%. Insulin like growth factor-1, dexamethasone, dibutyryl cAMP and phorbol 12-myristate 13-acetate, all of which are modulators of aromatase in other tissues, had no significant effect on aromatase activity in LNCaP cells. In addition, the cAMP-dependent protein kinase and protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine (H-7) had no effect on the enzyme activity. Factors affecting prostatic aromatase may be distinct from those for other known species.
Cancer Lett 1996 Apr 19
PMID:Modulation of aromatase activity by growth factors in an androgen sensitive human prostate cancer cell line, LNCaP. 860 65

A quantitative polymerase chain reaction (PCR) method for determining concentrations of mRNA for the cyclic AMP (cAMP)-binding protein RI alpha, a regulatory subunit of cAMP-dependent protein kinase, was developed using site-directed mutagenic primers and mix-melt PCR. The PCR product for RI alpha mRNA was modified to include an EcoRV restriction site for use as an internal standard. This mutant utilised the same primers as the target mRNA and differed in sequence by only four bases. As only one of these base changes results in a purine/pyrimidine switch the effective change in labelling with [32P]dCTP was less than 0.5%. Reverse transcription of mRNA was performed and quantitative PCR was carried out using fixed levels of mutant RI alpha vs varying amounts of both normal RI alpha sequence of known concentration and unknown samples. Validation of the technique using rigourous quality control established that reverse transcription, determined by incorporation of labelled nucleotides, gave intra- and interassay variations of 16.2 and 9.3% respectively. Using crossover evaluation of cDNA concentrations with cloned RI alpha sequences as controls intra- and interassay variations of 14.3% and 4-8% respectively were obtained. Using compounded errors, the limits of precision for this technique demonstrate that values that are altered by 50% or more represent a true alteration in mRNA levels between samples tested. This value compares favourably to similar values for radioimmunoassays of between 10% and 30% precision. Analysis of a series of patient samples during routine follow-up of treatment demonstrated clear changes in mRNA levels. Using site-directed mutagenesis to establish a quantitative PCR-based assay for expression of mRNA this study demonstrates the potential usefulness and some limitations of quantitative PCR for applications within a clinical biochemistry laboratory. However, based on compounded error, values that vary by less than 50% within assays, and by less than 70% in separate assays could not be clearly separated. Assessment of paired patient samples has demonstrated clear changes in mRNA for the target protein RI alpha. With the use of normal quality control procedures this study has established that the degree of reproducibility of this quantitative PCR technique would allow assessment of mRNA levels for markers of interest in clinical samples in a routine laboratory context.
Br J Cancer 1996 Jun
PMID:Analysis of cAMP RI alpha mRNA expression in breast cancer: evaluation of quantitative polymerase chain reaction for routine use. 866 26

A phosphorylation site was introduced into chimeric monoclonal antibody B72.3 (MAb-chB72.3) by site-specific mutation of the coding sequence. The phosphorylation site for the cAMP-dependent protein kinase was positioned at the carboxyl terminus of the heavy-chain constant region of MAb-chB72.3. The resultant modified MAb-chB72.3-P was expressed in 293 cells and purified. The MAb-chB72.3-P protein was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chB72.3-P protein bound to cells expressing the tumor-associated glycoprotein 72 antigen. The introduction of phosphorylation sites into MAbs provides a new type of MAb for the diagnosis and treatment of cancers.
Cancer Res 1996 Sep 15
PMID:Construction of phosphorylatable monoclonal antibody to a tumor-associated antigen. 879

Drug resistance in cancer poses a major problem to the success of chemotherapy. Increased resistance to the DNA-damaging chemotherapeutic drug cisplatin may be associated with a variety of factors including decreased drug accumulation, increased intracellular levels of thiols, and increased DNA repair. We have found that mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells harboring a defective regulatory subunit (RI) of the cAMP-dependent protein kinase (PKA) exhibited increased resistance to cisplatin. These mutants are cross-resistant to other DNA-damaging chemotherapeutic agents, including bleomycin and melphalan. In addition, wild-type CHO cells transfected with and overexpressing the yeast phosphodiesterase gene or a dominant mutant Rl alpha subunit gene also displayed similar increased resistance to cisplatin. However, mutants with altered catalytic (C) subunits showed a sensitivity to cisplatin similar to the wild-type cells. Further analysis by gel shift assay using cisplatin-damaged DNA as probes and nuclear extracts derived from the Rl subunit mutants showed increased binding of nuclear factor(s) to the damaged DNA. In addition, a host cell reactivation assay of DNA repair, using a cisplatin-damaged reporter plasmid, detected enhanced capacity for repair of DNA lesions in the PKA mutants. These results suggest that DNA repair may be increased in the PKA mutants. We speculate that functional inactivation of PKA may result in increased DNA repair and the acquisition of resistance to DNA-damaging anticancer drugs in cancer.
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PMID:Cisplatin resistance and regulation of DNA repair in cAMP-dependent protein kinase mutants. 885 7

Increased expression of the RI alpha subunit of cAMP-dependent protein kinase type I has been shown in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. The sequence-specific inhibition of RI alpha gene expression by an antisense oligodeoxynucleotide results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin. A single-injection RI alpha antisense treatment in vivo also causes a reduction in RI alpha expression and inhibition of tumor growth. Tumor cells behave like untransformed cells by making less protein kinase type I. The RI alpha antisense, which produces a biochemical imprint for growth control, requires infrequent dosing to restrain neoplastic growth in vivo.
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PMID:Protein kinase A-directed antisense restrains cancer growth: sequence-specific inhibition of gene expression. 891 9

Natural cytotoxicity (NC) against cancer involves receptor-ligand interactions between lymphohemopoietic cells that mediate NC against tumor cells. The only candidate for a receptor on cells mediating NC is NC-1.1, identified using mAb 1C4. In this study we showed that mAb 1C4 blocked NC-1.1+ cell conjugation to WEHI-164 tumor cells, indicating that NC-1.1 is a surface protein required for cell-cell interaction. Affinity-purified NC-1.1 was a 45-kDa monomeric protein. It was a good in vitro substrate for cyclic GMP (cGMP)-dependent protein kinase (PKG) and protein kinase C (PKC) and a relatively poor substrate for cAMP-dependent protein kinase (PKA). Phosphopeptide mapping revealed one phosphopeptide phosphorylated by PKG and PKA, and two additional peptides phosphorylated by PKC. Phosphorylation by PKG or PKA abolished phosphorylation at the PKC sites, while coincubation of NC-1.1 with both PKG and PKC reduced phosphorylation of all sites. NC-1.1 was also a phosphoprotein after immunoprecipitation from intact spleen cells and its phosphorylation was increased after cell stimulation with PKC or PKG activators (phorbol esters or 8-bromo-cGMP). The possible consequences of intracellular signaling were tested in functional assays for NC. Phorbol ester activation of spleen cells increased NC, while 8-bromo-cGMP and 8-bromo-cAMP had little effect. However, coincubation with both phorbol ester and either 8-bromo-cGMP or 8-bromo-cAMP virtually abolished NC without affecting cell conjugation. These results suggest that NC-1.1 is a receptor for a ligand on certain tumor cells and reveal that key intracellular signaling pathways involving PKC, PKG, and PKA interact to effect a coordinated control of NC.
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PMID:Phosphorylation of the NC-1.1 receptor and regulation of natural cytotoxicity by protein kinase C and cyclic GMP-dependent protein kinase. 903 46

The signal transduction pathway of cAMP, mediated by the cAMP-dependent protein kinase (PKA), is involved in the regulation of metabolisms, cell growth and differentiation and gene expression. Isolated PKA mutants from Chinese hamster ovary (CHO) cells were used in our laboratory to study the role of cAMP in the development of drug resistance in cancer. We have found that PKA mutants harboring a defective regulatory (RI alpha) subunit, but not the catalytic (C) subunit, are more resistant to the chemotherapeutic drug cisplatin. To clarify the role of PKA in cisplatin resistance, we have performed a step-wise selection with a CHO RI alpha subunit mutant cell line, 10248, for further resistance to cisplatin. A representative clone (10248/CDDP(R)-5) was used for further characterization. These cisplatin-resistant PKA mutant cells remained refractory to cAMP-induced growth inhibition and had decreased PKA activity comparable to the parental 10248 mutant cells. Furthermore, 10248/CDDP(R)-5 also exhibited cross-resistance to the nitrogen mustard melphalan but maintained the same sensitivity as wild-type cells to non-DNA-damaging agents such as methotrexate. The mechanism of resistance may be due to increased DNA repair as assessed by the host cell reactivation assay. We speculate that mutation and functional inactivation of PKA may result in deregulated growth response to cAMP, as well as the acquisition of resistance to cisplatin and other DNA-damaging agents in cancer.
Int J Cancer 1997 Jul 17
PMID:Characterization of a cAMP-dependent protein kinase mutant resistant to cisplatin. 921 44

Multidrug resistance (MDR) in cancer poses a major obstacle to the success of chemotherapy. We previously reported that cyclic AMP (cAMP)-resistant mutants of the Chinese hamster ovary and the mouse adrenal cortical carcinoma cells harboring defective regulatory (RI alpha) subunits of the cAMP-dependent protein kinase (PKA) are more sensitive than wild-type cells to chemotherapeutic agents that are substrates for P-glycoprotein. In addition, a transfectant overexpressing a mutant RI alpha cDNA showed similar increased sensitivity to these drugs. The altered drug sensitivity in the RI alpha mutants results from reduced expression of the mdr gene, suggesting that PKA may regulate its expression. In this study, we evaluated the sensitivity of several Chinese hamster ovary catalytic (C) subunit mutants to various anticancer drugs. Like the RI alpha subunit mutant, the C subunit mutants also exhibit decreased kinase activity and unresponsiveness to growth inhibition by cAMP. However, in contrast to the RI alpha subunit mutant, the C subunit mutants are not multidrug sensitive and maintain P-glycoprotein expression levels comparable to those of wild-type cells. Furthermore, the C subunit mutants display the same resistance patterns as wild-type cells to P-glycoprotein substrates, including Adriamycin, Taxol, and colchicine. No significant difference was observed in their sensitivity to non-MDR drugs, such as 5-fluorodeoxyuridine, between wild-type, RI alpha, and C subunit mutant cells. These results suggest that the increased multidrug sensitivity in the PKA mutant cells results from alteration of the RI alpha subunit and not the kinase activity, thus implying novel functions for the RI alpha subunit. Therefore, genetic alteration of the RI alpha subunit of PKA may modulate drug resistance in cancer.
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PMID:Regulation of P-glycoprotein expression in cyclic AMP-dependent protein kinase mutants. 941 12

Cathepsin B (CB), a lysosomal cysteine proteinase, is implicated in cancer metastasis and inflammatory tissue injury. We examined the effects of the protein kinase agonists and inhibitors on the regulation of CB activity in THP-1 human monocytic cells by two macrophage activators, lipopolysaccharide (LPS) and interferon- (IFN- ). CB elevation induced by LPS alone or LPS followed by IFN- was blocked by protein kinase C (PKC) inhibitors staurosporine, H-7, phloretin and bisindolylmaleimide, and by cyclic nucleotide-dependent protein kinase inhibitors HA 1004, H-8, H-89 and cAMP-dependent protein kinase (PKA) inhibitor. The CB activity by LPS and IFN- were augmented by diacylglycerol kinase inhibitor. PKC activator, phorbol 12-myristate 13-acetate (PMA) and PKA activator, dibutyryl cAMP could replace LPS in priming the cells for IFN- stimulation but 8-bromo-cGMP did not. These findings suggest that the activation of PKC and PKA appears to be involved at least in part in the induction of CB activity in THP-1 cells.
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PMID:Effect of protein kinase modulators on the regulation of cathepsin B activity in THP-1 human monocytic leukemia cells. 945 27

Peptide growth factors regulate normal cellular proliferation and differentiation through autocrine and paracrine pathways and are involved in cancer development and progression. Among the endogenous growth factors, the epidermal growth factor (EGF)-related proteins play an important role in the pathogenesis of human cancer. In fact, overexpression of EGF-related growth factors such as transforming growth factor alpha and amphiregulin and/or their specific receptor, the EGF receptor (EGFR), has been detected in several types of human cancers, including breast, lung, and colorectal cancers. Therefore, the blockade of EGFR activation by using anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The cAMP-dependent protein kinase (PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth and differentiation. Two PKA isoforms with identical catalytic (C) subunits but different cAMP-binding regulatory (R) subunits (defined as RI in PKAI and RII in PKAII) have been identified. Predominant expression of PKAII is found in normal nonproliferating tissues and in growth-arrested cells, whereas enhanced levels of PKAI are detected steadily in tumor cells and transiently in normal cells exposed to mitogenic stimuli. Overexpression of PKAI has been correlated recently with poor prognosis in breast cancer patients. Inhibition of PKAI expression and function by specific pharmacological agents such as the selective cAMP analogue 8-chloro-cAMP (8-Cl-cAMP) induces growth inhibition in various human cancer cell lines in vitro and in vivo. We have provided experimental evidence of a functional cross-talk between ligand-induced EGFR activation and PKAI expression and function. In fact, PKAI is overexpressed and activated following transforming growth factor alpha-induced transformation in several rodent and human cell line models. Furthermore, PKAI is involved in the intracellular mitogenic signaling following ligand-induced EGFR activation. We have shown that an interaction between EGFR and PKAI occurs through direct binding of the RI subunit to the Grb2 adaptor protein. In this respect, PKAI seems to function downstream of the EGFR, and experimental evidence suggests that PKAI is acting upstream of the mitogen-activated protein kinase pathway. We have also demonstrated that the functional interaction between the EGFR and the PKAI pathways could have potential therapeutic implications. In fact, the combined interference with both EGFR and PKAI with specific pharmacological agents, such as anti-EGFR blocking MAbs and cAMP analogues, has a cooperative antiproliferative effect on human cancer cell lines in vitro and in vivo. The antitumor activity of this combination could be explored in a clinical setting because both the 8-Cl-cAMP analogue and the anti-EGFR blocking MAb C225 have entered human clinical trial evaluation. Finally, both MAb C225 and 8-Cl-cAMP are specific inhibitors of intracellular mitogenic signaling that have different mechanisms of action compared with conventional cytotoxic drugs. In this respect, a cooperative growth-inhibitory effect in combination with several chemotherapeutic agents in a large series of human cancer cell lines in vitro and in vivo has been demonstrated for anti-EGFR blocking MAbs or for 8-Cl-cAMP. Therefore, the combination of MAb C225 and 8-Cl-cAMP following chemotherapy could be investigated in cancer patients.
Clin Cancer Res 1998 Apr
PMID:Interactions between the epidermal growth factor receptor and type I protein kinase A: biological significance and therapeutic implications. 956 74


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