The Presence Of 12-O-tetradecanoylphorbol-13-acetate Research Articles

Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation

Epigalloccatechin-3-gallate (EGCG) is the main polyphenol component of green tea (leaves of Camellia sinensis). EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a key role in the processes of extracellular matrix (ECM) remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and tumor necrosis factor alpha (TNF-α) in human retinal pigment epithelial cells (HRPECs). EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2) by inhibiting generation of reactive oxygen species (ROS). EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs). Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31) on corneal neovascularization (CNV) induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.

Contribution of reactive oxygen species to migration/invasion of human glioblastoma cells U87 via ERK-dependent COX-2/PGE2 activation

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation, an increase in the migration/invasion of U87 glioblastoma cells was detected by a wound healing assay, transwell analysis, and spheroid formation assay by inducing matrix metalloproteinase-9 (MMP-9) enzyme activity via a gelatin zymographic analysis. A dose- and time-dependent increase in cyclooxygenase-2 (COX-2) gene expression with elevated prostaglandin E2 (PGE2) production was identified in TPA- but not in 4α-TPA (a respective inactive compound)-treated U87 cells TPA-induced migration/invasion was significantly blocked by adding the COX-2-specific inhibitor, NS398, through a reduction in PGE2 production. Data from the pharmacological studies using specific chemical inhibitors showed that activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) was involved in TPA-induced migration/invasion, COX-2 protein expression, and MMP-9 activation. Stimulation of intracellular peroxide production by TPA was detected by a DCHF-DA assay, and the addition of superoxide dismutase (SOD) or tempol significantly inhibited TPA-induced migration/invasion and COX-2 protein expression accompanied by a decrease in peroxide production. An increase in NADPH oxidase activity by TPA was examined, and TPA-induced migration/invasion was blocked by adding DPI, an NADPH oxidase inhibitor. Additionally, the natural flavonoids quercetin (QE), baicalein (BE), and myricetin (ME) effectively blocked TPA-induced migration/invasion while simultaneously inhibiting COX-2/PGE2 production, MMP-9 enzyme activity, and peroxide production in U87 cells. The contribution of ROS production to the migration/invasion of U87 glioblastoma cells via ERK-activated COX-2/PGE2 and MMP-9 induction was first investigated here, and agents such as QE, BE, and ME with the ability to block these events possess the potential to be developed for use against migration/invasion by glioblastomas.

N-terminal Truncation of Antiapoptotic MCL1, but Not G2/M-induced Phosphorylation, Is Associated with Stabilization and Abundant Expression in Tumor Cells

The antiapoptotic BCL2 family member MCL1 is normally up- and down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK- and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G2/M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK- and GSK3 (but not G2/M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death.

Geldanamycin, a heat-shock protein 90-binding agent, induces thymocyte apoptosis through destabilization of Lck in presence of 12-O-tetradecanoylphorbol 13-acetate

Geldanamycin, a heat-shock protein 90 (Hsp90)-binding agent, modulates various cellular activities. The present study found that, although geldanamycin by itself had no effect on thymocyte viability, it induced apoptosis in thymocytes with a reduction of the mitochondrial transmembrane potential (DeltaPsim) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC). This apoptosis depended on transcription and translation, and on activation of caspase-8 and -3. Geldanamycin treatment in the presence of TPA also enhanced destabilization of Lck. This destabilization was independent of transcription and translation. It was inhibited, however, by conventional PKC inhibitors, preventing apoptosis. Proteasome inhibitor affected neither the degradation of Lck nor DNA fragmentation, although they inhibited reduction of DeltaPsim. These results suggest that the ubiquitin-proteasome system is not involved in Lck destabilization, and that DeltaPsim reduction is not directly related to the progression of apoptosis. Furthermore, inhibition of Lck in the presence of TPA induced apoptosis in thymocytes. Our findings suggest that Hsp90 modulates thymocyte apoptosis in concert with PKC through the destabilization of Lck and in a caspase-8- and -3-dependent manner.

The Evi1 proto‐oncoprotein blocks endomitosis in megakaryocytes by inhibiting sustained cyclin‐dependent kinase 2 catalytic activity

The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA-stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid-specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1-expressing, TPA-treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNA's, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA-stimulated Evi1-expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.

Chronic effects of ethanol on muscarinic acetylcholine receptors are modulated by protein kinase C

We have previously demonstrated that long-term ethanol treatment increased the number and function of muscarinic acetylcholine receptors (mAChRs) in human neuroblastoma cells, but the molecular mechanisms involved in these changes are unknown. In the present study, the effect of protein kinase C (PKC) on these events was investigated in human neuroblastoma SH-SY5Y cells. Following exposure to 100 mM ethanol for 2 days, both [³H]N-methylscopolamine binding and carbachol-stimulated I(1,4,5)P₃formation were increased. When cells were cultured in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of PKC, the effects of ethanol on mAChR number were totally inhibited but ethanol still potentiated carbacholstimulated I(1,4,5)P₃ formation in TPA treated cells. TPA dose-dependently inhibited carbochol-stimulated I(1,4,5)P₃ formation and this effect appeared to be independent of PKC phosphorylating activity. On the other hand, PKC inhibitors mimicked ethanol effects on mAChR number and function. Selective inhibition of classical PKC isozymes with 1 μΜ Gö 6976 for 2 days caused an increase in mAChR number and function, suggesting a role for these isozymes in ethanol-induced upregulation of mAChRs. These data indicate that longterm ethanol treatment may upregulate the number of mAChRs by counteracting PKC-mediated phosphorylation. The effects of ethanol on receptor-coupled phosphoinositide hydrolysis appear to be independent of PKC activity.

Activation of human herpesvirus 8 (HHV-8) thymidine kinase (TK) TATAA-less promoter by HHV-8 ORF50 gene product is SP1 dependent.

Human herpesvirus 8 (HHV-8) is a newly discovered virus closely associated with Kaposi's sarcoma and primary effusion lymphomas. When they occur in patients with AIDS, these B-cell lymphomas frequently harbor another human herpesvirus, Epstein-Barr virus (EBV). To determine the molecular mechanisms of the regulation of early gene expression by the immediate-early gene products of HHV-8 and to assess possible molecular interactions between HHV-8 and EBV, we studied the regulation of the HHV-8 thymidine kinase (TK) promoter in cell lines harboring either or both viruses. The constitutive chloramphenicol acetyltransferase (CAT) activity of the TK promoter was low in all six cell lines tested. A putative immediate-early gene product of HHV-8 ORF50, which is a homolog of EBV BRLF1, was cloned into an expression vector and tested for its transactivating capacity. In the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the CAT activity of the TK promoter was increased 7- to 720-fold by cotransfection with the ORF50 clone in EBV-producing cell lines (Ramos/AW, P3HR-1, and BC-1) but not in EBV-negative cell lines (BCBL-1 and Ramos), nor in the latently EBV-infected cell line Raji. The TK promoter contains three consensus SP1- and two AP1-binding sites. In electrophoretic mobility shift assays, the cellular factor SP1, but not AP1, was found to bind specifically to the TK promoter. To determine whether the increased CAT activity resulted from the interaction of SP1 with the ORF50 gene product, we introduced mutations into two SP1-binding sites. Both mutated SP1 sites had reduced SP1-binding activity and greatly decreased TK promoter responsiveness to ORF50 transactivation, suggesting that upregulation of TK promoter by ORF50 is SP1 dependent.

Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-alpha production in RBL-2H3 cells.

To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells. Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used. Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4). All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01). These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.

Ecotropic C-type retrovirus of B16 melanoma and malignant transformation of normal melanocytes.

We reported previously that B16,JB/RH and JB/MS melanomas of C57BL/6 mice express the common melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody (MAb). This MAA is encoded by the env gene of an ecotropic MuLV-type retrovirus that somatically emerged in melanomas of C57BL/6 mice. The potential role of this melanoma-associated retrovirus (MelARV) in melanoma formation remains unknown and has not been previously investigated. To test this, normal melanocyte lines (melan-a and C57M) of C57BL/6 mice were infected with the MelARV produced by B16BL6 melanoma. Infection of these melanocytes with the MelARV was associated with the appearance of the MAA recognized by MM2-9B6 MAb. Most of the infected melanocyte sublines were able to grow only in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Two infected melanocyte sublines showed morphological changes, were able to grow in the absence of TPA and, after inoculation into C57BL/6 mice, produced rapidly growing, highly pigmented tumors. These new melanomas, derived from the MelARV-infected melan-a and C57M melanocytes, were termed Meli-A1 and Meli-BL, respectively. Southern blot analysis of EcoRI- and HindIII-digested DNAs from these melanomas showed several retroviral insertion sites. One copy of MeIARV was found to be inserted at the end of the 6th leucine domain of the c-maf proto-oncogene, which encodes a basic region/leucine zipper transcription factor related to the AP-1 family that is able to form homodimers or heterodimers with Fos and Jun transcription factors. Our data indicate that c-maf is a common insertion site of MelARV in BL6, Meli-A1 and Meli-BL melanomas, whereas no such insertion site was found in the melanocytes infected with MelARV but not malignantly transformed. Thus, our data imply that the ecotropic MelARV that somatically emerged in B16 and other melanomas of C57BL/6 mice may play a role in malignant transformation.

Ecotropic C‐type retrovirus of B16 melanoma and malignant transformation of normal melanocytes

We reported previously that B16, JB/RH and JB/MS melanomas of C57BL/6 mice express the common melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody (MAb). This MAA is encoded by the env gene of an ecotropic MuLV-type retrovirus that somatically emerged in melanomas of C57BL/6 mice. The potential role of this melanoma-associated retrovirus (MelARV) in melanoma formation remains unknown and has not been previously investigated. To test this, normal melanocyte lines (melan-a and C57M) of C57BL/6 mice were infected with the MelARV produced by B16BL6 melanoma. Infection of these melanocytes with the MelARV was associated with the appearance of the MAA recognized by MM2-9B6 MAb. Most of the infected melanocyte sublines were able to grow only in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Two infected melanocyte sublines showed morphological changes, were able to grow in the absence of TPA and, after inoculation into C57BL/6 mice, produced rapidly growing, highly pigmented tumors. These new melanomas, derived from the MelARV-infected melan-a and C57M melanocytes, were termed Meli-A1 and Meli-BL, respectively. Southern blot analysis of EcoRI- and HindIII-digested DNAs from these melanomas showed several retroviral insertion sites. One copy of MelARV was found to be inserted at the end of the 6th leucine domain of the c-maf proto-oncogene, which encodes a basic region/leucine zipper transcription factor related to the AP-1 family that is able to form homodimers or heterodimers with Fos and Jun transcription factors. Our data indicate that c-maf is a common insertion site of MelARV in BL6, Meli-A1 and Meli-BL melanomas, whereas no such insertion site was found in the melanocytes infected with MelARV but not malignantly transformed. Thus, our data imply that the ecotropic MelARV that somatically emerged in B16 and other melanomas of C57BL/6 mice may play a role in malignant transformation. Int. J. Cancer 76:430–436, 1998.© 1998 Wiley-Liss, Inc.

Effects of steroid hormones and cyclosporine A on taurine-transporter activity in the RAW264.7 cell line.

The activity of the taurine transporter is affected by various extracellular stimuli, such as ions, hormones and stress. To assess the effects of steroid hormones and cyclosporine A (CsA) on taurine-transporter activity, the murine monocytic cell line, RAW264.7, was stimulated with dexamethasone (DM), triamcinolone (TA), cortisone (CS), hydrocortisone (HCS), prednisone (PSN), prednisolone (PSL) and methylprednisolone (MPSL) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of the cell with TPA led to a significant reduction in taurine-transporter activity. However, in the case of the stimulation of the cells with steroid hormones in the presence of TPA, all of the hormones reversed the TPA-induced reduction in the taurine-transporter activity. Treatment of the cells with CsA led to a significant reduction in taurine-transporter activity, but ionomycin (IM) alone did not affect taurine-transporter activity. However, IM reversed the TPA- and CsA-induced reduction in taurine-transporter activity. These results showed that both IM and the glucocorticoid hormones reversed TPA-induced reductions in taurine-transporter activity but only IM reversed the CsA-induced reduction of transporter activity in the RAW264.7 cell line.

Enhanced sensitivity to tumor growth and development in multistage skin carcinogenesis by transforming growth factor-alpha-induced epidermal growth factor receptor activation but not p53 inactivation.

Transforming growth factor-alpha (TGF alpha) can stimulate keratinocyte proliferation and function as an autocrine tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated TGF alpha-transgenic mouse skin. In this study, we examined the effect of ectopic TGF alpha transgene expression on skin tumor growth and progression after DMBA initiation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the multiplicity and size of skin tumors arising in TGF alpha-transgenic mice were significantly higher than those of the nontransgenic parental CD-1 strain. There were more dysplastic papillomas and squamous cell carcinomas (SCCs) in the transgenic animals as well. ProTGF alpha protein was expressed in transgenic papillomas, but mature TGF alpha was not detected. The epidermal growth factor receptor (EGFR) appeared to be downregulated and was associated with enhanced tyrosine phosphorylation of several substrates in TGF alpha-transgenic mouse tumors. Characteristic codon 61 mutations in the Ha-ras gene were found in most of the papillomas and SCCs induced by DMBA and TPA in transgenic as well as nontransgenic mice. However, no p53 gene mutations were found in any skin tumors from either transgenic or control animals. Analysis of cellular proliferation in both DMBA-TPA-induced papillomas and in skin 48 h after TPA treatment alone revealed significantly more DNA synthesis in TGF alpha-transgenic mice relative to controls. These results demonstrate that TGF alpha, through EGFR overstimulation, can act synergistically with TPA to induce the formation, growth, and development of DMBA-initiated skin tumors containing classic Ha-ras gene mutations but not p53 gene inactivation.

Effects of three different Ca(2+)-ATPase inhibitors on Ca2+ response and leukotriene release in RBL-2H3 cells.

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

Modulation of GM-CSF receptor beta-subunit and interleukin-6 receptor mRNA expression in a human megakaryocytic leukemia cell line.

Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor beta-subunit (GM-CSFR beta-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR beta-subunit mRNA was expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR beta-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de novo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while IL-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of beta 1, beta 3, alpha 5, alpha v, and alpha IIb subunits of the integrin family in spreading human erythroleukemia cells

The localization of five integrin subunit proteins was studied in human erythroleukemia (HEL) cells spreading on various culture substrata in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and the absence of serum. The cells readily adhered on fibronectin, but TPA was needed for adherence on vitronectin and for the spreading of the cells on both substrata. Indirect immunofluorescence microscopy showed that in the spread cells cultured on vitronectin or fibronectin for 2 hours, beta 1, beta 3, alpha 5, and alpha IIb integrin subunits were localized at focal adhesions as identified by talin-immunoreactivity. The alpha v integrin immunoreactivity was initially found at the focal adhesions when the cells were cultured on vitronectin, but was also found later in cells cultured on fibronectin. The alpha IIb integrin immunoreactivity disappeared from focal adhesions within 24 hours. The alpha 5 and beta 1 integrin immunoreactivities disappeared from the focal adhesions in cells cultured on vitronectin, but not in cells cultured on fibronectin. When the cells were plated on glass substratum in the presence of TPA, they spread much slower than on vitronectin or fibronectin, but some cells showed focal adhesions after only 8 hours in culture. In this case, the alpha v and beta 3 integrin subunits were found at focal adhesions. After TPA treatment, HEL cells deposited thrombospondin-immunoreactive material onto their culture substratum, but synthesis of fibronectin, vitronectin, fibrinogen, or von Willebrand factor was not detected. Thus, the results suggest that TPA would activate several integrin receptors in HEL cells and also stimulate the secretion of thrombospondin, which might be used as an adhesion ligand for the integrin vitronectin receptor alpha v/beta 3 complex.

Localization of β1, β3, α5s, αv, and αIIb Subunits of the Integrin Family in Spreading Human Erythroleukemia Cells

Abstract The localization of five integrin subunit proteins was studied in human erythroleukemia (HEL) cells spreading on various culture substrata in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and the absence of serum. The cells readily adhered on fibronectin, but TPA was needed for adherence on vitronectin and for the spreading of the cells on both substrata. Indirect immunofluorescence microscopy showed that in the spread cells cultured on vitronectin or fibronectin for 2 hours, beta 1, beta 3, alpha 5, and alpha IIb integrin subunits were localized at focal adhesions as identified by talin-immunoreactivity. The alpha v integrin immunoreactivity was initially found at the focal adhesions when the cells were cultured on vitronectin, but was also found later in cells cultured on fibronectin. The alpha IIb integrin immunoreactivity disappeared from focal adhesions within 24 hours. The alpha 5 and beta 1 integrin immunoreactivities disappeared from the focal adhesions in cells cultured on vitronectin, but not in cells cultured on fibronectin. When the cells were plated on glass substratum in the presence of TPA, they spread much slower than on vitronectin or fibronectin, but some cells showed focal adhesions after only 8 hours in culture. In this case, the alpha v and beta 3 integrin subunits were found at focal adhesions. After TPA treatment, HEL cells deposited thrombospondin-immunoreactive material onto their culture substratum, but synthesis of fibronectin, vitronectin, fibrinogen, or von Willebrand factor was not detected. Thus, the results suggest that TPA would activate several integrin receptors in HEL cells and also stimulate the secretion of thrombospondin, which might be used as an adhesion ligand for the integrin vitronectin receptor alpha v/beta 3 complex.

Phorbol Ester and Interferon-γ Modulation of Epidermal Growth Factor Receptors on Human Amniotic (WISH) Cells

Abstract In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.

Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture.

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Platelet glycoprotein IIb/IIIa complex in cultured cells. Localization in focal adhesion sites in spreading HEL cells

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.

Platelet Glycoprotein IIb/IIIa Complex in Cultured Cells. Localization in Focal Adhesion Sites in Spreading HEL Cells

A panel of mouse monoclonal antibodies (MoAbs) was raised that react with platelet glycoproteins (GP) IIb or IIIa. On immunofluorescence, the MoAbs reacted with 30% to 40% of the human erythroleukemia (HEL) cells. When the HEL cells were induced to spread on fibronectin in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), the MoAbs reacted with the focal adhesion sites. Only some of the GPIIIa MoAbs reacted with cells other than platelets, megakaryocytes, or HEL cells, and these showed focal adhesion sites in cultured human endothelial cells, fibroblasts, and epithelial cells from normal kidney tubules. They did not react, however, with transformed fibroblasts, fibrosarcoma cells, cultured cells from hypernephromas, or cultured human amnion epithelial cells. The results suggest that the platelet-type GPIIb/IIIa complex is only expressed in cells showing an ability to define megakaryoblastic differentiation. Localization of the GPIIb/IIIa complex at the induced focal adhesion sites in HEL cells and localization of the GPIIIa-like molecules in other cells suggest their direct role in the adhesion process and in the actomyocin organization of adherent cells.