Tissue polypeptide antigen liberation and proliferation of MCF-7 cells after synchronization with hydroxytamoxifen and rescue with estradiol

The kinetic of release of the tissular polypeptide antigen (TPA) by MCF-7 cells synchronised by sequential treatment with hydroxytamoxifen (OH-TAM) and 17 beta-estradiol has been studied. The present findings confirm the proliferative effect of estradiol on MCF-7 cells, with a shortening of the doubling time (TD) (22.2 h versus 24.8 h) and an increase in the growing fraction (Fc) (94% versus 81%) when compared with the same parameters measured in cells rescued from OH-TAM but not treated with estradiol. In addition, the action of estradiol was followed by a simultaneous increase in the amount of TPA in the culture medium related with the phases G2/M and G1 of the mitotic cycle. This phenomenon seems to be the reason for the steplike shape of the TPA released curves. The experimental results suggest that in MCF-7 cells the sequential combination of antiestrogenic agents and estrogens leads to a cellular synchronization. Furthermore this synchronization is maintained for at least 3 cycles of cell division.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor-mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), affects RPE cell adhesion in a substratum-dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long-lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long-term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell-cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long-term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long-term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC. © 1993 Wiley-Liss, Inc.     

Regulation of the shape and size of human erythrocytes by protein kinases A and C

The effect of 12-O-tetradecanoyl-phorbol-12-acetate (TPA) and dibutyryl-3,5-cAMP on the shape, volume and protein phosphorylation in human red blood cells (RBC) was studied. TPA (but not cAMP) reduced the average volume of RBC and their transformed definite pool in the cup-shaped form. The changes in the physical parameters were accompanied by an increase in the cytoskeleton protein phosphorylation. An additive effect of cAMP and TPA on the phosphorylation of bands 4.1 and 4.9 was established, thus indicating that distinct domains of amino acid residues were phosphorylated by these stimuli. It was concluded that protein kinase C regulates the shape and volume of human RBC. A model, in which the state of spectrin 4.1-actin and actin 4.9-myosin complexes define the shape and volume of RBC and thus influence ion transport, is proposed.     

不同类型甜瓜成熟过程中果肉质地及其细胞显微结构的变化

采用质地剖面分析(TPA)和穿刺方法,测定3种不同口感质地的5个甜瓜材料(梗硬果肉的P10和3-6、脆酥果肉的417和20-5以及软果肉的Charentais)不同成熟时期果肉硬度、咀嚼性和黏着性以及脆性和平均硬度,评价甜瓜果肉的质地特性;采用组织切片法观察果肉组织细胞的显微结构,并通过Image-pro plus 6.0软件测定细胞大小参数(细胞面积、周长、长度及宽度)和形状参数(细胞纵横比及圆度),明确不同果肉质地类型甜瓜果实成熟过程中果肉细胞显微结构的变化特征,探讨甜瓜细胞形态参数与果肉质地的关系,为甜瓜品质育种提供理论依据。结果显示:(1)梗硬果肉甜瓜(P10和3-6)的果肉细胞较小,排列紧密;脆酥果肉甜瓜(417和20-5)的细胞较大,排列较疏松;软果肉甜瓜(Charentais)的细胞最大,排列极不规则。(2)不同口感甜瓜果肉在成熟期的细胞面积和周长差异显著,与口感呈显著负相关关系;在成熟过程中,甜瓜果肉细胞面积、周长和长度等表现出不同程度的增大,而细胞纵横比和圆度总体表现为下降趋势,即细胞越来越圆。(3)甜瓜果肉的口感与质地及细胞大小参数呈显著或极显著相关关系;细胞大小与黏着性呈显著或极显著正相关关系(0.951*~0.983**),细胞面积与TPA硬度及脆性呈显著负相关关系(分别为-0.910*和-0.926*),长度和宽度与脆性呈显著负相关关系(分别为-0.884*和-0.894*);细胞形状参数中的圆度与黏着性具有显著相关性(0.936*)。研究表明,口感不同的甜瓜果肉具有显著不同的质地和细胞显微结构,且甜瓜果肉口感与其果肉质地及细胞大小密切相关,即细胞越小,甜瓜果肉质地越硬。     

Constitutive Production of 92-kDa Gelatinase B Can Be Suppressed by Alterations in Cell Shape

We have examined the effect that cell shape has on production of the 92-kDa gelatinase B, an enzyme of the matrix metalloproteinase family thought to contribute to the invasiveness of both normal and malignant cells. Using the agent poly(HEMA) and a human melanoma cell line that constitutively produces both the 72- and 92-kDa gelatinases, we have found that alteration in cell shape, that is, a change in cell "roundness," resulted in a specific loss of the constitutive production of the 92-kDa gelatinase B. To examine this phenomenon further, cells were treated with an inhibitor of actin polymerization, cytochalasin D. This treatment also resulted in a loss of 92-kDa gelatinase B production, provided the cells were treated with drug from the outset of the experiment. If the cells were allowed to attach and spread prior to drug exposure, no loss of 92-kDa gelatinase B production was observed. Similar to the poly (HEMA) results, cytochalasin D had little effect on production of the 72-kDa gelatinase A. Treatment with the tublin polymerization inhibitor colchicine had no effect on 92-kDa gelatinase B production, nor did growth of the cells as three-dimensional tumor spheroids, although an alteration in cell morphology was observed in both instances. This phenomenon was studied in another system, namely, HL-60 cells, which were induced to differentiate into macrophage-like cells in response to TPA treatment and consequently produce the 92-kDa gelatinase B. HL-60 cells treated with TPA and cytochalasin D failed to produce the 92-kDa gelatinase B. These results suggest that the 92-kDa gelatinase B can be regulated by alterations in cell shape but more specifically, by alterations in the organization of the actin cytoskeleton. Furthermore, the mechanism responsible for cell shape/actin cytoskeletal down-regulation of the 92-kDa gelatinase B may be common to many cell types competent to produce this enzymatic activity.     

Flow cytometry of TPA-induced changes in cells of chronic B-cell leukemias

Cells from 14 patients with chronic B cell leukemias were cultured for up to 7 days with TPA (160 nM) in order to induce maturation of the malignant cells. Five cellular parameters, which can be quantitated by flow cytometry were analyzed in such cultures. These parameters were cell size, cell cycle, RNA, neutral esterase activity and dye uptake in mitochondria. Cell size increased in 13/14 cases in TPA treated cells compared to control cells. Cell cycle analysis revealed a low percentage of cells in S and G2/M phase both in control and TPA-treated cultures of chronic B cell leukemias, while in cultures of peripheral blood mononuclear cells TPA caused a large increase of S and G2/M cells. Both in chronic B cell leukemias and in PBMC, TPA induced an increase of RNA staining and neutral esterase activity in all or most cultures. Furthermore the staining of mitochondria increased in most cases. In conclusion, multiple changes can be induced by TPA in chronic B cell leukemias without associated proliferation.     

Tumor promoter-induced disruption of junctional complexes in cultured epithelial cells is followed by the inhibition of cytokeratin and desmoplakin synthesis

The organization and synthesis of proteins involved in the formation and stabilization of desmosome-type junctions was investigated in cultured epithelial cells treated with a tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate (TPA]. In Madin-Darby bovine (MDBK) and canine (MDCK) kidney cell colonies, TPA induced a rapid disruption of desmosomes and marked alterations in cell morphology. Within 4-6 h after TPA treatment, cell shape changed from cuboidal to highly irregular, with some very long extensions that contained cytokeratin fibrils, and many flat lamellar protrusions which were devoid of cytokeratin fibrils. These morphological changes in both MDBK and MDCK cells were followed by a dramatic and coordinated inhibition in the synthesis of all cytokeratins, 14-24 h after the addition of TPA, but without a similar effect on the synthesis of vimentin, which is coexpressed in these cells. In contrast, in dense cultures of MDBK and MDCK cells the synthesis of cytokeratins and the organization of desmosomal contacts were not affected by TPA. In an epithelial cell line derived from the bovine mammary gland (BMGE-H) the synthesis of an acidic cytokeratin of 45 kD, which was previously shown to be synthesized at high levels only in dense cultures, was dramatically inhibited by TPA treatment. Cell-free in vitro translation assays with mRNA from control and TPA-treated cells also demonstrated a decrease in the synthesis of cytokeratins in response to TPA. The inhibition of cytokeratin synthesis after TPA treatment was paralleled by a decrease in the synthesis of a high molecular weight (HMW) desmoplakin protein, which was abundant in dense MDBK and BMGE-H cells. The results with TPA-treated cells are suggestive of a coordinated down-regulation in the synthesis of only those cytokeratins and of a desmoplakin which were shown to be regulated by the extent of cell-cell contact. Cytokeratin phosphorylation in TPA-treated cells was low and reflected the decrease in their total mass, suggesting that it was not altered by TPA treatment. The possible linkage between the regulation of synthesis and organization of proteins involved in desmosome formation is discussed.     

Possible involvement of reorganization of actin filaments, induced by tumor-promoting phorbol esters, in changes in colony shape and enhancement of proliferation of cultured epithelial cells

Tumor promoters are known to induce reorganization of actin, morphological changes and enhancement of proliferation of epidermal cells in vivo. In this study, we have examined the effects of tumor promoters on these events to clarify the role played by the organization of actin filaments in the regulation of the shape and growth of colonies of epithelial cells in culture. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a change in the shape of colonies of FL and Madin-Darby canine kidney (MDCK) cells within 6 hr. Changes in the shape of colonies were consistent with the morphological change of individual cells and the dissociation of groups of cells in the colonies. Addition of TPA also caused reorganization of actin filaments after 2 hr, and it caused enhancement of proliferation of FL and MDCK cells after 48 hr but did not cause any such changes in KB cells. However, the binding affinities of 4 beta-phorbol 12,13-dibutyrate (PDBu) to FL and MDCK cells were similar to that of PDBu to KB cells. Related tumor promoters such as phorbol 12,13 didecanoate (PDD) and mezerein caused effects similar to those caused by TPA. In contrast, nontumor promoting phorbol esters, such as 4 alpha-PDD and phorbol, had no effect. Cyclic AMP blocked the TPA-induced changes in FL and MDCK cells. These results suggest that TPA-induced reorganization of actin filaments which can be inhibited by cyclic AMP results in changes in the shape of colonies and enhancement of proliferation.     

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.     

Interleukin 1 and protein kinase C activator are dissimilar in their effects on Il-2 receptor expression and Il-2 secretion by T lymphocytes

T lymphocytes respond to mitogenic stimulation by expressing the receptor for interleukin 2 (Il-2) and secreting Il-2; once the receptor is expressed, Il-2 induces these cells to proliferation. In the present report using mouse T lymphocytes, thymocytes, and the lymphoma cell line EL4, we studied receptor expression and Il-2 secretion as early parameters for T-lymphocyte activation in response to ionomycin, concanavalin A (Con A), 12-O-tetradecanoyl-phorbol 13-acetate (TPA), and interleukin 1 (Il-1). Il-1 is required for mitogenic response of lymphocyte preparations that are rigorously depleted of macrophages. On its own, Il-1 had very little effect on Il-2 secretion and Il-2 receptor expression by T lymphocytes. TPA strongly synergized with ionomycin both for Il-2 secretion and for Il-2 receptor expression whereas Il-1 did not. Il-1 required the simultaneous presence of ionomycin and TPA to have any demonstrable effect on T lymphocytes from spleen and on thymocytes. However, on EL4 cells which were also partially responsive to TPA alone, Il-1 showed strong synergy with TPA to induce Il-2 secretion and Il-2 receptor expression. The effect of Il-1 on EL4 cells was dose dependent where increasingly higher concentrations of Il-1 in the presence of a fixed concentration of TPA caused higher percentage of EL4 cells to become Il-2 receptor positive. The present results suggest that Il-1 does not cause its effect on T lymphocytes via the same mechanism of protein kinase C activation that has been proposed for TPA.     

Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells

Prior studies showed that sphingomyelinase action and the free sphingoid bases inhibited protein kinase C (Kolesnick, R. N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). The present studies investigated whether sphingomyelinase action also inhibited a biologic process mediated via protein kinase C, phorbol ester-induced differentiation of HL-60 promyelocytic cells into macrophages. The potent phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated time- and concentration-dependent conversion of HL-60 cells into macrophages, ED50 congruent to 5 x 10(-10) M. Differentiation involved growth inhibition, adherence of the suspended cells to tissue culture plastic, morphologic changes, and development of specific enzymatic markers. Sphingomyelinase, which increased the level of sphingoid bases and inactivated protein kinase C, prevented this event. In control incubations, cell number increased 2.10-fold over 24 h, and 2 +/- 1% of the cells were adherent. In incubations with TPA (0.5 nM), cell number increased only 1.75-fold, and 30% were adherent. Sphingomyelinase (3.8 x 10(-5) unit/ml) restored growth to incubations containing TPA to 2.02-fold and reduced adherence to 15%. Sphingomyelinase (3.8 x 10(-2) unit/ml) also restored growth partially and reduced adherence to a maximal concentration of TPA (3 nM). Similar results were obtained with the sphingoid base sphingosine (3-4.5 microM). Sphingomyelinase antagonized the morphologic changes associated with conversion to the macrophage phenotype. Untreated HL-60 cells presented typical promyelocytic morphology with large nuclei, little cytoplasm, and uniformity of nuclear and cell shape. TPA induced a larger cell population with abundant cytoplasm and unusual shape. Sphingomyelinase prevented these changes. Sphingomyelinase blocked TPA-induced increases in the macrophage marker enzymes, acid phosphatase and alpha-naphthyl acetate esterase. These studies indicate that the action of a sphingomyelinase, like the sphingoid bases, blocks phorbol ester-induced differentiation of HL-60 cells into macrophages and provides further support for the concept that sphingomyelinase action may be sufficient to comprise a physiologically relevant inhibitory pathway for protein kinase C.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

TPA-induced signal transduction: a link between PKC and EGFR signaling modulates the assembly of intercellular junctions in Caco-2 cells

Recent studies suggest that signal transduction may have an important role in the development and regulation of the metastatic phenotype. Here, we investigated the role of the epidermal growth factor receptor (EGFR), and protein kinase C (PKC), in the process of reassembly of cadherin-dependent cell-cell adhesion of Caco-2 cells. We used chemical activation of PKC and EGFR with 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent, pretreatment with protein kinase inhibitors and subcellular fractionation to analyze the effect of the phorbol ester on the redistribution of junctional proteins. Transepithelial resistance (TER), electron microscopy and immunofluorescence analyses were also carried out. Activation with TPA resulted in disassembly of adherens junctions (AJs), but the tight junction (TJ) structure and function remained unaltered. TPA affected E-cadherin levels. In Caco-2 cells at day 2 of culture, when most E-cadherin is not associated with the cytoskeleton, a decrease in the level of this protein was observed as soon as 6 h after TPA addition. However, at day 5 of culture, the major effect observed after 6 h of treatment was a translocation of the protein from the Triton-insoluble to the -soluble fraction. On the other hand, TPA did not significantly affect the E-cadherin-associated proteins alpha and beta-catenins. Potent specific EGFR inhibitors, such as PD153035 and Tyrphostin 25, as well as Calphostin C, an inhibitor of PKC, significantly blocked the effect of TPA on AJs. Furthermore, inhibition of the TPA effect by the PD98059 MAPK inhibitor suggests that activation of this kinase was the final event in the modulation of cadherin-dependent cell-cell adhesion. Pretreatment of cell monolayers with Calphostin C before EGF treatment, one of the ligands of EGFR, blocked the redistribution of E-cadherin caused by EGF. Based on these results, we conclude that both EGFR and PKC activation are involved in TPA-induced cell signaling for modulation of cadherin-dependent cell-cell adhesion and cell shape in Caco-2 cells.     

Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.     

Differential effects of phorbol ester on the in vitro invasiveness of malignant and non-malignant human fibroblast cells

The effect of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI-38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3-fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI-38 cell invasion. Immunoblots demonstrated that in both cell lines the alpha and beta isoforms of protein kinase C were equally down-regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI-38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non-invasive phenotype.     

Phosphorylation and dephosphorylation of human promyelocytic leukemia cell (HL-60) proteins by tumor promoter

Human promyelocytic leukemia cell line (HL-60) has been shown to be induced to the terminal differentiation into macrophage-like cells by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The present studies describe the effects of TPA on the phosphorylation of HL-60 cell proteins. A rapid decrease in the phosphorylation of a 75 kD protein was observed within a few minutes after treatment with TPA. On the other hand, TPA treatment of HL-60 cells caused rapid increase in the phosphorylation of a 67 kD protein and other minor proteins. Phorbol and 4α-phorbol-12,13-dodecanoate, both of which are biologically inactive derivatives of TPA, failed to cause any changes in protein phosphorylation in HL-60 cells. These results suggest that changes in protein phosphorylation are involved in mechanisms of the differentiation in HL-60 cells induced by TPA. Cell fractionation experiments revealed that 67K protein was located in cytosol. Though 75K protein also seemed to be located in cytosol, the phosphate moiety of 75K protein was almost lost during cell fractionation, suggesting that the phosphorylation of 75K protein was specifically regulated in HL-60 cells. Dimethyl sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxy-vitamin D₃, all of which induce the differentiation in HL-60 cells, did not cause any changes in protein phosphorylation. These results suggest that the changes in protein phosphorylation are specific for TPA. The possible mechanisms of changes in protein phosphorylation by TPA were discussed.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)