Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recent findings that oestradiol and progestins activate the Src/Ras/Erks signalling pathway raise the question of the role of this stimulation. Microinjection experiments of human mammary cancer-derived cells (MCF-7 and T47D) with cDNA of catalytically inactive Src or anti-Ras antibody prove that Src and Ras are required for oestradiol and progestin-dependent progression of cells through the cell cycle. The antitumoral ansamycin antibiotic, geldanamycin, disrupts the steroid-induced Ras-Raf-1 association and prevents Raf-1 activation and steroid-induced DNA synthesis. Furthermore, the selective MEK 1 inhibitor, PD 98059, inhibits oestradiol and progestin stimulation of Erk-2 and the steroid-dependent S-phase entry. The MDA-MB231 cells, which do not express oestradiol receptor, fail to respond to oestradiol in terms of Erk-2 activation and S-phase entry. Fibroblasts are made equally oestradiol-responsive in terms of DNA synthesis by transient transfection with either the wild-type or the transcriptionally inactive mutant oestradiol receptor (HE241G). Co-transfection of catalytically inactive Src as well as treatment with PD98059 inhibit the oestradiol-dependent S-phase entry of fibroblasts expressing either the wild-type oestrogen receptor or its transcriptionally inactive mutant. The data presented support the view that non-transcriptional action of the two steroids plays a major role in cell cycle progression.
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PMID:Non-transcriptional action of oestradiol and progestin triggers DNA synthesis. 1022 64

A nonmetastatic human mammary epithelial cell line (MCF-10A) was engineered to overproduce protein kinase Calpha (PKCalpha) so as to investigate a role for this isoform in the metastatic phenotype. PKCalpha transfectants (clone 26alpha) expressed an 8-fold higher level of PKCalpha protein without compensatory alterations in other isoforms. Clone 26alpha proliferated slowly (accumulating in G1 of the cell cycle) but exhibited pronounced increases in motility and adhesion. Elevated expression of cell cycle inhibitor p27 and focal adhesion proteins was observed, whereas E-cadherin expression decreased to undetectable levels. These observations were consistent with the morphology of PKCalpha transfectants (large, disaggregated, and flat, with lamellipodia and extensive actin fibers) and control cells (small, aggregated, and refractile). Treatment with PKC inhibitors or transfection of a dominant negative (dn) mutant of Rac1, but neither dn RhoA nor dn cdc42, reduced the motility of clone 26alpha, implicating PKCalpha catalytic activity and endogenous Rac1, respectively, in the PKCalpha-induced phenotype. Overall, PKCalpha overexpression suppresses proliferation while endowing MCF-10A cells with properties consistent with the metastatic phenotype.
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PMID:Overexpression of protein kinase Calpha in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation, and motility. 1035 15

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.
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PMID:KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules. 1038 57

The enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type 1 (PKA-1) has been correlated with cancer cell growth. We have investigated the effects of sequence-specific inhibition of RI alpha gene expression on the growth of MCF-7 human breast cancer cells. We report that RI alpha antisense treatment results in a reduction in RI alpha expression at both mRNA and protein levels and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology, cleavage of poly(ADP-ribose) polymerase (PARP) and appearance of apoptotic nuclei. In addition, bcl-2 protein level was reduced and p53 expression increased in growth arrested cells. Interestingly, RI alpha antisense inhibited cell viability and induced apoptosis in the absence of p53, suggesting that these actions of RI alpha antisense are exerted independent of p53. In contrast, two- and four-base mismatched control oligonucleotides had no effect on either cell growth or morphology. These results demonstrate that the RI alpha antisense, which efficiently depletes the growth stimulatory molecule RI alpha, induces cell differentiation and apoptosis, providing a new approach to combat breast cancer cell growth.
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PMID:Growth arrest and induction of apoptosis in breast cancer cells by antisense depletion of protein kinase A-RI alpha subunit: p53-independent mechanism of action. 1039 66

Expression of the RIalpha regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RIalpha was replaced with Ser (RIalpha-p) for the structure-function analysis of RIalpha. MCF-7 hormone-dependent breast cancer cells were transfected with an expression vector for the wild-type RIalpha or mutant RIalpha-p. Overexpression of RIalpha-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RIalpha overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RIIbeta, suppressed RIalpha and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.
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PMID:Ala99ser mutation in RI alpha regulatory subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth. 1039 71

Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting. CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer.
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PMID:Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. 1040 94

Breast cancer is the most frequent cancer in women while it is the second cause of cancer death. Estrogens are well recognized to play the predominant role in breast cancer development and growth and much efforts have been devoted to the blockade of estrogen formation and action. The most widely used therapy of breast cancer which has shown benefits at all stages of the disease is the use of the antiestrogen Tamoxifen. This compound, however, possesses mixed agonist and antagonist activity and major efforts have been devoted to the development of compounds having pure antiestrogenic activity in the mammary gland and endometrium. Such a compound would avoid the problem of stimulation of the endometrium and the risk of endometrial carcinoma. We have thus synthesized an orally active non-steroidal antiestrogen, EM-652 (SCH 57068) and the prodrug EM-800 (SCH57050) which are the most potent of the known antiestrogens. EM-652 is the compound having the highest affinity for the estrogen receptor, including estradiol. It has higher affinity for the ER than ICI 182780, hydroxytamoxifen, raloxifene, droloxifene and hydroxytoremifene. EM-652 has the most potent inhibitory activity on both ER alpha and ER beta compared to any of the other antiestrogens tested. An important aspect of EM-652 is that it inhibits both the AF1 and AF2 functions of both ER alpha and ER beta while the inhibitory action of hydroxytamoxifen is limited to AF2, the ligand-dependent function of the estrogen receptors. AF1 activity is constitutive, ligand-independent and is responsible for mediation of the activity of growth factors and of the ras oncogene and MAP-kinase pathway. EM-652 inhibits Ras-induced transcriptional activity of ER alpha and ER beta and blocks SRC-1-stimulated activity of the two receptors. EM-652 was also found to block the recruitment of SRC-1 at AF1 of ER beta, this ligand-independent activation of AF1 being closely related to phosphorylation of the steroid receptors by protein kinase. Most importantly, the antiestrogen hydroxytamoxifen has no inhibitory effect on the SRC-1-induced ER beta activity while the pure antiestrogen EM-652 completely abolishes this effect, thus strengthening the need to use pure antiestrogens in breast cancer therapy in order to control all known aspects of ER-regulated gene expression. In fact, the absence of blockade of AF2 by hydroxytamoxifen could explain why the benefits of tamoxifen observed up to 5 years become negative at longer time intervals and why resistance develops to tamoxifen. EM-800, the prodrug of EM-652, has been shown to prevent the development of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat, a well-recognized model of human breast cancer. It is of interest that the addition of dehydroepiandrosterone, a precursor of androgens, to EM-800, led to complete inhibition of tumor development in this model. Not only the development, but also the growth of established DMBA-induced mammary carcinoma was inhibited by treatment with EM-800. An inhibitory effect was also observed when medroxyprogesterone was added to treatment with EM-800. Uterine size was reduced to castration levels in the groups of animals treated with EM-800. An almost complete disappearance of estrogen receptors was observed in the uterus, vaginum and tumors in nude mice treated with EM-800. EM-652 was the most potent antiestrogen to inhibit the growth of human breast cancer ZR-75-1, MCF-7 and T-47D cells in vitro when compared with ICI 182780, ICI 164384, hydroxytamoxifen, and droloxifene. Moreover, EM-652 and EM-800 have no stimulatory effect on the basal levels of cell proliferation in the absence of E2 while hydroxytamoxifen and droloxifene had a stimulatory effect on the basal growth of T-47D and ZR-75-1 cells. EM-652 was also the most potent inhibitor of the percentage of cycling cancer cells. (ABSTRACT TRUNCATED)
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PMID:EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium. 1041 81

The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a 'biologic' anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327 --> Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.
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PMID:Sex hormone-binding globulin, its membrane receptor, and breast cancer: a new approach to the modulation of estradiol action in neoplastic cells. 1041 27

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.
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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

The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the physiologically active form of vitamin D3 that inhibits proliferation and induces differentiation of a variety of malignant cells. We evaluated a newly synthesized vitamin D3 analogue [1,25(OH)2-16-ene-5,6-trans-D3 (Ro 25-4020)] that has a novel 5,6-trans motif. Dose-response studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 had 10-100-fold greater antiproliferative activities than 1,25(OH)2D3 when measuring clonal growth of breast (MCF-7) and prostate (LNCaP) cancer cell lines as well as a myeloid leukemia cell line (HL-60). Because the chief toxicity of vitamin D3 is hypercalcemia, we examined the calcemic activity of 1,25(OH)2-16-ene-5,6-trans-D3 in mice. Remarkably, 1,25(OH)2-16-ene-5,6-trans-D3 was at least 40-fold less calcemic as compared with 1,25(OH)2D3 and 1,25(OH)2-16-ene-D3 (Ro 24-2637). To explore the mechanism by which the 1,25(OH)2-16-ene-5,6-trans-D3 analogue mediated its antiproliferative activity, several studies were performed. Pulse-exposure studies showed that a 4-day pulse exposure to 1,25(OH)2-16-ene-5,6-trans-D3 (10(-7) M) in liquid culture was adequate to achieve a 40% inhibition of MCF-7 clonal growth in the absence of the analogue, suggesting that the growth inhibition mediated by 1,25(OH)2-16-ene-5,6-trans-D3 was at least in part irreversible. Cell cycle studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 increased the proportion of MCF-7 cells in the G0-G1 phase and decreased those in the S phase. Furthermore, 1,25(OH)2-16-ene-5,6-trans-D3 induced an elevated expression of the cyclin-dependent kinase inhibitors, p21waf1 and p27kip1. In addition, 1,25(OH)2-16-ene-5,6-trans-D3 almost completely inhibited telomerase activity, as measured by telomeric repeat amplification protocol assay and human telomerase reverse transcriptase mRNA. For each of the growth-related parameters that were examined, the vitamin D3 analogue was more active than 1,25(OH)2D3. In contrast, 1,25(OH)2D3 was more calcemic than 1,25(OH)2-16-ene-5,6-trans-D3. In summary, 1,25(OH)2-16-ene-5,6-trans-D3, having a novel 5,6-trans motif, strongly inhibited clonal proliferation and reduced telomerase activity with low calcemic activity, suggesting further testing in in vivo cancer models. This analogue may gain a therapeutic niche for selected malignancies.
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PMID:5,6-trans-16-ene-vitamin D3: a new class of potent inhibitors of proliferation of prostate, breast, and myeloid leukemic cells. 1046 2


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