Tumor-promoting Phorbol Esters Research Articles

Microtubule-dependent subcellular redistribution of the transcriptional coactivator p/CIP.

The transcriptional coactivator p/CIP is a member of a family of nuclear receptor coactivator/steroid receptor coactivator (NCoA/SRC) proteins that mediate the transcriptional activities of nuclear hormone receptors. We have found that p/CIP is predominantly cytoplasmic in a large proportion of cells in various tissues of the developing mouse and in a number of established cell lines. In mouse embryonic fibroblasts, serum deprivation results in the redistribution of p/CIP to the cytoplasmic compartment and stimulation with growth factors or tumor-promoting phorbol esters promotes p/CIP shuttling into the nucleus. Cytoplasmic accumulation of p/CIP is also cell cycle dependent, occurring predominantly during the S and late M phases. Leptomycin B (LMB) treatment results in a marked nuclear accumulation, suggesting that p/CIP undergoes dynamic nuclear export as well as import. We have identified a strong nuclear import signal in the N terminus of p/CIP and two leucine-rich motifs in the C terminus that resemble CRM-1-dependent nuclear export sequences. When fused to green fluorescent protein, the nuclear export sequence region is cytoplasmic and is retained in the nucleus in an LMB-dependent manner. Disruption of the leucine-rich motifs prevents cytoplasmic accumulation. Furthermore, we demonstrate that cytoplasmic p/CIP associates with tubulin and that an intact microtubule network is required for intracellular shuttling of p/CIP. Immunoaffinity purification of p/CIP from nuclear and cytosolic extracts revealed that only nuclear p/CIP complexes possess histone acetyltransferase activity. Collectively, these results suggest that cellular compartmentalization of NCoA/SRC proteins could potentially regulate nuclear hormone receptor-mediated events as well as integrating signals in response to different environmental cues.

Isozyme-specific abnormalities of PKC in thyroid cancer: evidence for post-transcriptional changes in PKC epsilon.

PKC isozymes are the major binding proteins for tumor-promoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKCalpha at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKCepsilon was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of PKCepsilon or alpha. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKCalpha 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKCepsilon. However, 8 of 11 PCs had major isozyme-specific reductions of the PKCepsilon protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKCepsilon are highly prevalent in thyroid tumors and may play a significant role in their development.

Isozyme-Specific Abnormalities of PKC in Thyroid Cancer: Evidence for Post-Transcriptional Changes in PKC Epsilon

PKC isozymes are the major binding proteins for tumor-promoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKCα at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKCε was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of PKCε or α. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKCα 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKCε. However, 8 of 11 PCs had major isozyme-specific reductions of the PKCε protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKCε are highly prevalent in thyroid tumors and may play a significant role in their development.

Effect of arsenic on transcription factor AP-1 and NF-κB DNA binding activity and related gene expression

Both acute (24 h) and chronic (10–20 week) exposure of human fibroblast cells to low dose sodium arsenite (As(III)) significantly affects activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) DNA binding activity. Short-term treatment with 0.1–5 μM As(III) up-regulates expression of c-Fos and c-Jun and the redox regulators, thioredoxin (Trx) and Redox factor-1 (Ref-1) and activates both AP-1 and NF-κB binding. Chronic exposure to 0.1 or 0.5 μM As(III) decreased c-Jun, c-Fos and Ref-1 protein levels and AP-1 and NF-κB binding activity, but increased Trx expression. Short term exposure to phorbol 12-myristate 13-acetate (TPA), a phorbol ester tumour promoter, or hydrogen peroxide (H 2O 2) also activates AP-1 and NF-κB binding. However, pre-treatment with As(III) prevents this increase. These results suggest that As(III) may alter AP-1 and NF-κB activity, in part, by up-regulating Trx and Ref-1. The different effects of short- versus long-term As(III) treatment on acute-phase response to oxidative stress reflect changes in the expression of Ref-1, c-Fos and c-Jun, but not Trx.

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca2+ increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.

Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor

The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, β-phorbol 12,13-didecanoate (β-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of β-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with β-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with β-PDD induced pro-MBP mRNA and protein expression within 6 h. β-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of β-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor

The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A's proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, beta-phorbol 12,13-didecanoate (beta-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of beta-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with beta-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with beta-PDD induced pro-MBP mRNA and protein expression within 6 h. beta-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of beta-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

Stimulation of progesterone production in human granulosa-lutein cells by lipoproteins: evidence for cholesterol-independent actions of high-density lipoproteins.

Low-density lipoproteins (LDL) have been consistently reported to stimulate ovarian steroidogenesis, apparently by the provision of cholesterol as a steroidogenic substrate. Recent studies suggest that high-density lipoproteins (HDL) can also deliver cholesterol to support progesterone synthesis in human granulosa-lutein cells. Therefore, this study investigated the contributions of (i) cholesterol delivery, (ii) cyclic AMP and (iii) protein kinase C (PKC) in the steroidogenic responses of human granulosa-lutein cells to HDL and LDL. Over a 24-h treatment incubation, HDL stimulated a larger increase in progesterone output than did LDL at equivalent cholesterol concentrations. Moreover, at equal protein concentrations (100 microg protein/ml), HDL doubled progesterone production by cells co-treated with a maximally effective concentration of 22R-hydroxycholesterol, whereas LDL had no effect on the progesterone response to this membrane-permeable sterol. These observations indicate that the progesterone response to HDL is not solely due to the delivery of cholesterol as a steroidogenic substrate. Over 24 h, the stimulation of progesterone synthesis by HDL was additive with the response to a maximally effective concentration of dibutyryl-cAMP, but was unaffected by the down-regulation of PKC activity (by chronic pre-treatment with a tumour-promoting phorbol ester). We have concluded that HDL appears to stimulate progesterone production in human granulosa-lutein cells by a mechanism not solely reliant on cholesterol delivery.

Novel "nonkinase" phorbol ester receptors: the C1 domain connection.

In recent years, there have been great advances in our understanding of the pharmacology and biology of the receptors for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG). The traditional view of protein kinase C (PKC) as the sole receptor for the phorbol esters has been challenged with the discovery of proteins unrelated to PKC that bind phorbol esters with high affinity, suggesting a high degree of complexity in the signaling pathways activated by DAG. These novel "nonkinase" phorbol ester receptors include chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), and Munc13 isoforms (scaffolding proteins involved in exocytosis). In all cases, phorbol ester binding occurs at the single C1 domain present in these proteins and, as in PKC isozymes, ligand binding is a phospholipid-dependent event. Moreover, the novel phorbol ester receptors are also subject to subcellular redistribution or "translocation" by phorbol esters, leading to their association to different effector and/or regulatory molecules. Clearly, the use of phorbol esters as specific activators of PKC in cellular models is questionable. Alternative pharmacological and molecular approaches are therefore needed to dissect the involvement of each receptor class as a mediator of phorbol ester/DAG responses.

Overexpression of a constitutively active form of c-src in skin epidermis increases sensitivity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

Transgenic mice were developed to study the role of c-src in epithelial tumorigenesis through targeted expression of a constitutively active form of murine c-src (src(529)). Src(529) was targeted to the interfollicular epidermis with the human keratin 1 (HK1) promoter. The skin phenotype of these mice was characterized by exaggerated epidermal hyperplasia and hyperkeratosis within the first week after birth. The severity of this phenotype correlated with overall src kinase activity, both of which subsided with age. Treatment of adult HK1.src(529) transgenic mice with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate resulted in an increase in epidermal hyperplasia and labeling index significantly greater than that seen in nontransgenic littermates. In addition, HK1.src(529) transgenic mice developed papillomas earlier and in significantly greater numbers compared with nontransgenic littermates in a standard initiation-promotion experiment. The data support the hypothesis that activation of c-src kinase plays a role in skin tumor promotion.

Influence of 50 Hz electromagnetic fields in combination with a tumour promoting phorbol ester on protein kinase C and cell cycle in human cells.

It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogenesis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the family of protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levels including the activation of transcription factors, we evaluated in the present study the effects of exposure of human amniotic fluid cells (AFC) to 50 Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity, and on the cell cycle distribution. Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the membrane fraction after exposure to 10, 50, 100 nM, and 1 microM TPA. EMF exposure alone showed no effect on PKC translocation. Co-exposure to 10, 50, and 100 nM TPA and I mT EMF revealed a significant additive effect (25 +/- 50, 66 +/- 29, 22 +/- 50%, respectively) with the most prominent increase at the concentration of 50 nM TPA. At the highest concentration of TPA used (1 microM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity, showing a significant increase of 10 +/- 16% in total PKC activity after co-exposure to 50 nM TPA and 1 mT EMF when compared to 50 nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G0/G1-phase followed by a delayed transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that in AFC cells TPA at lower concentrations (< or = 100 nM) induces a less than maximum effect on the PKC pathway, which can be enhanced by the applied EMF.

Role of Ca2+ and protein kinase C in cholinergic, and alpha1-adrenergic agonists and EGF stimulated mitogen-activated protein kinase activity in lacrimal gland.

The MAP kinase pathway plays a key regulatory role in a variety of cellular functions including cell growth and differentiation, and gene expression.1,2 This pathway is characterized by a family of protein-serine/threonine kinases known as mitogen-activated protein kinases or MAPKs. Activation of MAPK is mediated by several mechanisms including the stimulation of G-protein coupled receptors (GPCR), such as muscarinic and angiotensin II receptors, or the activation of growth factor receptors via a protein-tyrosine kinase pathway.3,4 Protein kinase C (PKC), induced by phorbol ester tumor promoters, can activate the MAPK pathway in a Ras-independent manner.5 In addition, Ca2+ and cAMP-dependent pathways intersect with the MAPK signaling pathway, either stimulating or inhibiting it in different types of cells.6 Mechanisms of transactivation of epidermal growth factor (EGF) receptors in an EGF-independent manner, can mediate GPCR activation of MAPK.7 KeywordsContact LensCholesterol EsterLipid LayerCompositional CharacteristicNonpolar LipidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane for treating corneal diseases with total limbal stem cell deficiency.

Stem cells (SC) for the corneal epithelium are located exclusively at the limbus, i.e., the anatomic junction between the cornea and conjunctiva. Limbal epithelial SC are the ultimate source of regeneration of the entire corneal epithelium under normal and injured states. The epithelial phenotype of the limbal basal epithelium does not express corneal epithelial-specific keratin3, keratin 12 and connexin 43. As shown in cell-cycle kinetic studies, some portions of the limbal basal epithelial cells are slow-cycling and label-retaining. Other studies further confirmed limbal epithelial SC have greater growth potential in explant cultures and higher clonogenicity when cocultured on 3T3 fibroblast-feeder layers than corneal epithelial cells, and their proliferative potential is resistant to tumor-promoting phorbol esters.

プロテインキナーゼＣのシステインリッチドメインの化学合成，機能解析とドラッグデザイン

Protein kinase C (PKC) isozymes play a critical role in many signal transduction pathways and are also main targets of tumor-promoting phorbol esters. Conventional and novel PKC isozymes contain two cysteine-rich C 1 domains (C 1 A and C 1 B), both of which are candidates for phorbol ester binding sites. These C 1 domains of about 50-70 amino acids were synthesized by an Fmocsolid phase strategy, and were successfully folded by zinc treatment. We measured the dissociation constants (Kd's) of [3H] phorbol-12, 13-dibutyrate for all PKC C 1 peptides. Most of the C 1 peptides showed strong PDBu binding affinities with Kd's in the nanomolar range (0.45-7.4 nM) comparable to the respective whole PKC isozymes. The resultant C 1 peptide library can be used to screen for new ligands with PKC isozyme and importantly C 1 domain selectivity. We have recently developed a new lactone analogue of benzolactams (6) which shows significant selectivity in the PKCη-C 1 B binding on the basis of the structure-activity relationship of teleocidin-type tumor promoters.

Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy.

Recent years have seen extensive growth in the understanding of the role(s) of the various PKC isozymes and novel receptors for the phorbol ester tumor promoters. The PKC family of serine-threonine kinases is an important regulator of signaling cascades that control cell proliferation and death, and therefore represent targets for cancer therapy. While past interests have focused on PKC-selective inhibitors, more recently, intensive research has been underway for selective activators and inhibitors for each individual PKC isozyme. In the past few years a large number of PKC activators and inhibitors with potential as anticancer agents have been developed. A number of these compounds are already in Phase II clinical testing. As a new generation of cancer chemotherapeutic agents are designed, developed and put through a series of rigorous clinical trials, we can anticipate achieving exquisite control over PKC-mediated regulatory pathways, leading ultimately to a greater understanding of different cancers.

Diacylglycerol Kinase γ Is One of the Specific Receptors of Tumor-Promoting Phorbol Esters

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (Kd's). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGKγ because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGKγ translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGKγ (DGKγ-C1A and DGKγ-C1B) were synthesized and tested for their PDBu binding along with whole DGKγ (Flag-DGKγ) expressed in COS-7 cells. DGKγ-C1A and Flag-DGKγ showed strong PDBu binding affinity, while DGKγ-C1B was completely inactive. Scatchard analysis of DGKγ-C1A and Flag-DGKγ gave Kd's of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGKγ is C1A. This is the first evidence that DGKγ is a specific receptor of tumor-promoting phorbol esters.

Involvement of p-15INK4b and p-16INK4a Gene Expression in Saikosaponin a and TPA-Induced Growth Inhibition of HepG2 Cells

Saikosaponin a, a purified ingredients of Chinese herb with known antitumor activity can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16INK4a and p-15INK4b in HepG2 were greatly induced by saikosaponin a while that of p-21CIP, p-27KIP and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16INK4a and p-15INK4b mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15INK4a and p-16INK4b gene expression and might be mediated by PKC signaling pathway.

Inhibition by costunolide of phorbol ester-induced transcriptional activation of inducible nitric oxide synthase gene in a human monocyte cell line THP-1

Costunolide, the predominant sesquiterpene lactone in Saussureae radix, has been reported to exhibit potent chemopreventive effects on carcinogenesis. Effects of costunolide on cellular activation induced by a tumor-promoting phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) were investigated using a reporter gene assay which was designed to reflect the promoter activity of the inducible nitric oxide synthase (iNOS) gene in a human monocyte cell line THP-1. iNOS promoter-dependent reporter gene activity was significantly increased by TPA, and the TPA-induced increase of the reporter gene activity was efficiently reduced by costunolide, with an IC 50 of approximately 2 μM. The addition of sulfhydryl (SH) compounds effectively abrogated the inhibitory effects of costunolide, suggesting the involvement of its reactivity with SH groups of target proteins and/or thiol-depleting property. The present findings may further explain the cancer-preventive property of costunolide.

The regulatory role of the C-terminal domain of connexin43.

The C-terminal (CT) domain of connexin43 (Cx43) is thought to be important in the control of gap junction function via: a.) CT phosphorylation-dependent control of gap junction assembly and gating, b.) interactions of CT with key regulatory binding partners. To more closely examine CT-dependent regulation, we have expressed a hemagglutinin-Cx43CT (amino acids 235-382) fusion protein in Normal Rat Kidney (NRK) cells under a tetracycline-responsive inducible promoter. Western blot analysis shows that Cx43CT expression is markedly induced by at least 48 h oftreatment with the tetracycline analogue, doxycycline. Furthermore, Cx43CT is modified within the cell, as several treatments/conditions that increase endogenous Cx43 phosphorylation induced a mobility shift in Cx43CT. Treatment with kinase activators, including epidermal growth factor (EGF) and the tumor promoting phorbol ester 12-O-tetradecanylphorbol-13-acetate (TPA), caused a shift in the mobility of the Cx43CT in a manner consistent with the mobility shift observed upon increased phosphorylation of endogenous Cx43. Similarly, Cx43CT in mitotic cells is extensively shifted, consistent with reports which show that Cx43 is phosphorylated to a unique phosphoisoform in mitotic cells. These results indicate that the Cx43CT can interact with at least some of the kinases that phosphorylate endogenous Cx43 in cells and possibly modulate the effects of kinase activation on gap junctional communication.

Phorbol ester tumour promoter mediated altered expression and regulation of matrix metalloproteinase-2 in a H-ras transformed cell line capable of benign tumour formation.

The matrix metalloproteinases (MMPs) are thought to play key roles in tumour formation and malignant progression. The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to the phorbol ester tumour promoter, PMA, in a H-ras transformed cell line, NR3, which is capable of benign tumour formation. PMA treatment of NR3 cells resulted in decreased expression of MMP-2 mRNA levels. Following a lag period, an accompanying change in gelatinolytic activity was also found. These PMA-mediated alterations in MMP-2 mRNA levels were independent of de novo protein synthesis and involved both transcriptional and post-transcriptional events. Most notably, PMA regulates MMP-2 mRNA expression through a mechanism involving message de-stabilization. Additionally, protein kinase C mediated events were found to play a role(s) in the regulation of MMP-2 message expression in NR3 cells. This study demonstrates several novel aspects regarding the regulation of MMP-2 expression in a H-ras transformed cell line and thereby provides further insight into the altered growth regulatory programs associated with H-ras mediated cellular transformation.