Treatment With 12-O-tetradecanoylphorbol-13-acetate Treatment Research Articles

Aurora kinase A induces migration and invasion by inducing epithelial-to-mesenchymal transition in colon cancer cells

Aurora kinase is a family of serine/threonine kinases intimately associated with mitotic progression and the development of human cancers. Studies have shown that aurora kinases are important for the protein kinase C (PKC)-induced invasion of colon cancer cells. Recent studies have shown that aurora kinase A promotes distant metastasis by inducing epithelial-to-mesenchymal transition (EMT) in colon cancer cells. However, the role of aurora kinase A in colon cancer metastasis remains unclear. In this study, we investigated the effects of aurora kinase A on PKC-induced cell invasion, migration, and EMT in human SW480 colon cancer cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) changed the expression levels of EMT markers, increasing α-SMA, vimentin, and MMP-9 expression and decreasing E-cadherin expression, with changes in cell morphology. TPA treatment induced EMT in a PKC-dependent manner. Moreover, the inhibition of aurora kinase A by siRNAs and inhibitors (reversine and VX-680) suppressed TPA-induced cell invasion, migration, and EMT in SW480 human colon cells. Inhibition of aurora kinase A blocked TPA-induced vimentin and MMP-9 expression, and decreased E-cadherin expression. Furthermore, the knockdown of aurora kinase A decreased the transcriptional activity of NF-κB and AP-1 in PKC-stimulated SW480 cells. These findings indicate that aurora kinase A induces migration and invasion by inducing EMT in SW480 colon cancer cells. To the best of our knowledge, this is the first study that showed aurora kinase A is a key molecule in PKC-induced metastasis in colon cancer cells.

Characterization of the human zinc finger nfx‑1‑type containing 1 encoding ZNFX1 gene and its response to 12‑O‑tetradecanoyl‑13‑acetate in HL‑60 cells.

Human promyelocytic HL‑60 cells can be differentiated into macrophage‑like cells by treatment with 12‑O‑tetra decanoylphorbol‑13‑acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)‑encoding genes contain duplicated GGAA motifs, which are frequently found in the TPA‑responding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCH‑type containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx‑1‑type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiple‑cloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100‑bp region positively responded to TPA. In addition, reverse transcription‑quantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL‑60 cells. These results indicated that expression of the TPA‑inducible ZNFX1 gene, which belongs to the group of interferon‑responsive genes, is regulated by the cis‑action of the duplicated GGAA motif.

Abstract 2106: Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation

Abstract Twist1 is a basic helix-loop-helix transcription factor and is known to play a role in tumor progression through regulation of genes involved in epithelial-mesenchymal transition. Twist1 is a known transcriptional target of signal transducer and activator of transcription-3 (Stat3). In previous studies, Stat3 was shown to play a role in skin tumor progression in the two-stage skin carcinogenesis model, at least in part, by regulating the levels of Twist1. Recent studies using keratinocyte-specific knockout (KO) of Twist1 indicate that Twist1 regulates keratinocyte proliferation during skin tumor promotion. Western blot analysis of lysates from both Twist1 deficient mouse primary keratinocytes and from epidermis isolated from BK5.Cre x Twist1flox/flox mice indicated that Twist1 KO leads to reduced levels of the cell cycle proteins Cyclin E1, E2F1, and Cdk2 and increased expression of p21 following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). ChIP analysis of epidermal lysates from BK5.Cre x Twist1flox/flox mice revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc, indicating direct transcriptional regulation of these cell cycle regulators. Moreover, Twist1 KO in keratinocytes impeded cell cycle progression by reducing the number of cells that advanced to S-phase. Further analyses have demonstrated that deletion of Twist1 either in primary keratinocytes or in vivo leads to an upregulation of p53, which is responsible for the increased expression of p21 observed in these cells. Keratinocyte specific deletion of Twist1 in vivo suppressed epidermal proliferation induced by TPA treatment compared to that observed in wild-type (WT) mice. To further characterize the potential role of Twist1 during epithelial carcinogenesis, we analyzed the impact of Twist1 deletion in vivo on bulge region keratinocyte stem cells (KSCs). Keratinocyte specific deletion of Twist1 in vivo led to a significant reduction in the number of label-retaining cells as well as the number of α6-integrin+/CD34+ cells in the hair follicles of untreated mice. An ongoing two-stage skin carcinogenesis experiment shows that BK5.Cre x Twist1flox/flox mice have significantly reduced tumor development as observed by decreased tumor multiplicity and delayed latency compared WT mice. Furthermore, tumors that developed in BK5.Cre x Twist1flox/flox mice have significantly reduced size compared to tumors on WT mice. Collectively, these findings suggest that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by RP140108, CA76520, and T32 ES007247. Citation Format: Jaya Srivastava, Okkyung Rho, John DiGiovanni. Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2015-2106

Lysine-specific Demethylase 1 Represses THP-1 Monocyte-to-macrophage Differentiation

To investigate the role of lysine-specific demethylase 1 (LSD1) in the process of THP-1 monocyte-to-macrophage differentiation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 (IL-6) in THP-1 monocytes and THP-1-derived macrophages. Chromatin immunoprecipitation (ChIP) assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation. IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChIP assay in LSD1-knockdown THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 0, 4, 8, 12, and 24 hours. Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP-1 monocytes. The expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation (P<0.01). LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation. With knockdown of LSD1, H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points (all P<0.05, except 24 hours). The percentage of macrophages increased significantly in the THP-1 cells with LSD1 knockdown (P<0.05). LSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells. Suppression of LSD1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.

Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger

Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H(2)O(2)) to H(2)O and O(2). In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H(2)O(2) and O(2) without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H(2)O(2) using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H(2)O(2) are significant events for monocyte/macrophage differentiation by TPA.

Changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases during the differentiation of human promyelocytic leukemia cell line HL-60

The metabolism of poly(ADP-ribose) is known to play important roles in the nuclear function of the mammalian cells. In this study, changes in the activities and gene expressions of poly(ADP-ribose) glycohydrolases (PARG) in HL-60 cells treated with 12- O-tetradecanoyl-phorbol-13-acetate (TPA) or a PARG inhibitor, tannic acid, were investigated. Nuclear PARG activities of HL-60 cells treated with TPA were reduced to 30–40% of the activity in untreated cells at 24 h, while PARG activities in the cytoplasm remained unchanged. The transient decrease in the nuclear PARG activity by TPA treatment was accompanied by differentiation as measured by the nitroblue tetrazolium (NBT) reducing activity and adhesion to the culture dishes. In the presence of H7, an inhibitor of protein kinase C (PKC), both the decrease in nuclear PARG activity and the induction of differentiation by TPA treatment were suppressed. On the other hand, treatment with tannic acid caused the nuclear PARG activity to decrease continuously while the NBT reducing activity increased, but no morphological differentiation to macrophage-like cells was apparent. In order to analyze PARG gene expression, we isolated the human PARG cDNA by the RT-PCR technique. RT-PCR analysis revealed that TPA treatment leads to a reduction in the PARG gene expression prior to the phenotypic expression of macrophage-like cell differentiation, which was diminished by the presence of H7. Also, PARG gene expression was reduced by tannic acid treatment. These results provide the first evidence that a transient decrease in nuclear PARG activity is important for the onset of differentiation of HL-60 cells to macrophage-like cells.

617 Urokinase-type plasminogen activator is transcriptionally repressed during 12-o-tetradecanoylphorbol-13-acetate-dependent differentiation of hl-60 cells

We have previously shown in NIH 3T3 fibroblasts that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or fibroblast growth factor-2 (FGF-2) activates the Ras/Erk signaling pathway in NIH 3T3 fibroblasts, leading to the induction of the urokinase-type plasminogen activator (uPA) gene. In this study, we characterize cis-acting elements involved in this induction. DNase I hypersensitive (HS) site analysis of the uPA promoter showed that two regions were enhanced after TPA and FGF-2 treatment. One was located 2.4 kb upstream of the transcription start site (−2.4 kb), where a known PEA3/AP1 (AGGAAATGAGGTCAT) element is located. The other was located in a previously undefined far upstream region. Sequencing of this region revealed a similar AP1/PEA3 (GTGATTCACTTCCT) element at −6.9 kb corresponding to the HS site. Deletion analysis of the uPA promoter in transient transfection assays showed that both PEA3/AP1 elements are required for full inducibility, suggesting a synergism between the two elements. When the two sites were inserted together upstream of a minimal promoter derived from the thymidine kinase gene, expression of the reporter gene was more strongly induced by TPA and FGF-2 than with either of the two elements alone. Alone, the −6.9 element was more potent than the −2.4 element. The involvement of AP1 as well as Ets transcription factors was confirmed by examining different promoter constructs containing deletions in either the AP-1 or the PEA3 element, and by using an expression plasmid for dominant negative Ets-2. Electromobility shift analyses using specific antibodies showed that c-Jun and, JunD bind to both elements with or without induction. In addition, ATF-2 binds to the −2.4-kb element even without induction and c-Fos to the −6.9-kb element only after induction. Accordingly, overexpression of c-Fos caused induction from the −6.9-kb element, but reduced induction from the −2.4-kb element. The involvement of the Ets-2 transcription factor was shown by using expression plasmids for wild-type and dominant negative Ets-2.

Selective roles of protein kinase C isoforms on cell motility of GT1 immortalized hypothalamic neurones.

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in beta-catenin, a cell-cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green fluorescence protein (EGFP)-tagged actin to monitor the dynamic rearrangement of filamentous-actin (F-actin) in living cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while safingol (a PKC alpha-selective inhibitor) blocked the TPA-induced lamellipodial actin structure. Both wound-healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with safingol or rottlerin (a PKC delta-selective inhibitor) clearly blocked this TPA effect, indicating that both PKC alpha and PKC delta may be positive regulators of neuronal migration. By contrast, PKC gamma-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKC gamma may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130Cas, a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130Cas was induced in a PKC alpha-dependent manner. Together, these results demonstrate that PKC alpha promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130Cas and FAK.

Similar effects of electroporational stress and treatment with the phorbol ester 12- O-tetradecanoylphorbol-13-acetate on vimentin expression in mouse plasmacytoma cells

In mouse plasmacytoma cells (MPC-11), an activation of the normally repressed vimentin gene was observed as a response to transfectional stress. Effects of electroporation on vimentin gene expression were compared at the cellular and chromatin level to those caused by treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA). At the cellular level, similar changes in vimentin gene activity and cell-cycle distribution were observed by flow cytometry, whereas at the chromatin level similar changes in patterns of hypersensitive regions were detected by DNase I mapping. Additionally, a region located 700 bp upstream of the transcriptional start became hypersensitive to DNase I digestion upon electroporation and TPA treatment. This region overlaps two adjacent AP-1-like binding elements and generates specific DNA/AP-1 complexes in bandshift experiments. Therefore, the transcription factor AP-1 seems to play a central role in the activation of vimentin gene expression induced by these 2 different forms of stress.

A binding protein to the DNase I hypersensitive site II in HLA-DR alpha gene was identified as NF90.

We previously observed that IFN gamma-inducible expression of the human MHC class II, HLA-DR alpha, gene was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) only in human monocytic leukemia THP-1 cells, but not in HeLa cells. In the HLA-DR alpha gene, three DNase I hypersensitive sites (DHS) are known to be present in the promoter region (DHS-I) and first intron (DHS-II and -III) and are assumed to be involved in HLA-DR alpha gene regulation. In this study, we found a binding factor which recognized a unique palindrome sequence (DHS-22) in the region of the DHS II site of the HLA-DR alpha gene in THP-1 cells and HeLa cells. The binding activity of this factor was decreased by TPA treatment in THP-1 cells, but not in HeLa cells. This binding activity was also detectable in nuclear extracts of bovine brains. Thus, we isolated the DHS-22 binding factor from bovine brain nuclear extracts and finally identified it as NF90 on the basis of molecular mass analysis of Lys-C-digested fragments and amino acid sequences of the two peptides of the trypsin-digested binding protein. The DHS-22 binding protein(s) in THP-1 cells is (are) further confirmed by reactivity to an antibody against NF90, and we have demonstrated that the GST fusion protein of NF90 interacts with DHS-22 by electrophoretic gel mobility shift assay (EMSA). The mRNA of NF90 was decreased by TPA treatment in THP-1 cells but not in HeLa cells. These results suggest that the binding of NF90 to the DNase I hypersensitive site II of HLA-DR alpha gene seems to negatively regulate HLA-DR alpha gene expression.

Regulation of Motility and Protease Expression in PKC-Mediated Induction of MCF-7 Breast Cancer Cell Invasiveness

We investigated a potentially central role of protein kinase C (PKC) in controlling multiple pathways in breast cancer cell invasiveness. To do this we evaluated the ability of pharmacologic agents that alter PKC activity to regulate the behavior of the poorly invasive human breast cancer cell line MCF-7. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a dramatic induction of the invasiveness of these cells (18-fold), an effect that concurrent treatment with the PKC inhibitor Bryostatin-1 was able to block. To characterize alterations in the cellular properties that might be responsible for these effects we measured the impact of these two agents on a number of processes thought to be important for invasiveness. The motility of the cells was first examined; it was markedly increased by treatment with TPA (20-fold) and again, Bryostatin-1 inhibited this stimulation. We next examined the expression of MMP-1, 3, 9, 10, and 11 (matrix metalloproteinases), all of which have been shown to be PKC responsive in other systems. We found that the expression and secretion of MMP-9 were increased by at least 100-fold, though all of the enzyme secreted was in the latent form. Finally, the expression of both urokinase plasminogen activator (UPA) and its receptor (UPAR) were induced after TPA treatment by 8- and 7-fold, respectively. In conclusion, we have shown that stimulation of PKC activity markedly increases the invasiveness of MCF-7 cells, and that this change in behavior is correlated with a coordinated set of biochemical and cellular changes which are likely to contribute to this process. These data highlight the possible utility of PKC inhibitors such as Bryostatin-1 as anti-invasive and/or antimetastatic agents. Bryostatin-1 is currently in early clinical trials as an anticancer agent.

Analysis of the TPA regulatory element in the genomic poly(ADP-ribose) synthetase gene in human leukemia U937 cells.

The human leukemia U937 cells differentiate into monocyte/macrophage-like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). We observed that during this process, both protein and mRNA levels for PARS markedly decreased in U937 cells. Through deletion analysis of the PARS regulatory gene, we found that the sequence within the first intron region was responsible for the TPA-dependent repression. Electrophoretic mobility shift assays (EMSAs) and Southwestern blot analysis indicate that this element bound specifically to a nuclear protein. TPA treatment abolished the binding of the protein in U937 cells but not in HeLa cells. DNase I footprinting data show that the cis regulatory element is located between residues 328 and 383. We further examined the function of this cis element (BS207) in a basal promoter regulatory reporter construct and found that this cis element (BS207) functions as an enhancer via the binding of an unknown trans-acting factor. TPA treatment diminished the binding activity of the factor in U937 cells, resulting in a decrease in the enhanced activity to the basal level. These results suggest that abolishment of the binding of a special nuclear protein to the first intron of the PARS gene is related to the TPA-responsive downregulation of PARS in U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA) downregulates expression of CD30 in erythroleukemia cell line K562.

CD30, a member of the tumour necrosis factor/nerve growth factor receptor superfamily, has been thought to have pleiotropic functions on immune response. However, there has been only a little information about the mechanism of CD30 expression. In this study, modulation of the CD30 molecule was investigated by the treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). When cultures were supplemented with TPA, CD30 transcript was downregulated in a dose- and time-dependent manner in the erythroleukemia cell line K562. Half reduction of CD30 transcript, precursor protein and surface protein was at 3 h, 6 h, and 40 h, respectively, by Northern blot and Western blot analyses. This consecutive reduction of both the transcript and proteins suggests that TPA directly inhibits the transcriptional step of CD30, and subsequently CD30 molecules would decrease on the cell surface. To determine whether the protein kinase C (PKC) pathway is involved in this reduction, a PKC inhibitor, 10 microM H-7, was added to the K562 culture. The addition of H-7 recovered the inhibitory effect of TPA, indicating that PKC is involved in the transcription of CD30. When either 2 micrograms/ml actinomycin D or 20 micrograms/ml cycloheximide was added simultaneously with TPA to the culture, the repressive effect of TPA on CD30 was abolished. These results showed that the repression would also partly involve ongoing mRNA and protein synthesis under TPA treatment.

Transient expression of M-CSF is important for osteoclast-like cell differentiation in a monocytic leukemia cell line.

Cells of U937, a human monocytic leukemia cell line, differentiate into macrophages by treatment with 12-o-tetradecanoylphorbol-13-acetate (TPA), whereas cells treated with 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] continue to grow without undergoing differentiation. When U937 cells were successively treated with TPA and 1,25-(OH)2D3, tartrate-resistant acid phosphatase-positive multinucleated cells appeared at 5 days after the treatment. These osteoclast-like cells released a soluble form of 45Ca from 45Ca-labeled bone particles. These cells were not formed when the order of treatment with TPA and 1,25-(OH)2D3 was reversed. Use of either dexamethasone or interferon-gamma (IFN-gamma) was effective in inhibiting the formation of these osteoclast-like cells. The expression of c-src, c-fms, and macrophage colony stimulating factor (M-CSF) was induced by TPA treatment; however, TPA-induced M-CSF gene transcription was attenuated by the subsequent addition of 1,25-(OH)2D3. Furthermore, both dexamethasone and IFN-gamma impaired the attenuation of M-CSF expression, suggesting that the transient expression of M-CSF may be important for the formation of osteoclast-like cells.

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells.

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of human plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2.

Chronic TPA treatment inhibits expression of proximal tubule-specific properties by LLC-PK1 cells.

Confluent LLC-PK1 cell populations expressed progressively proximal tubule-specific properties, including gamma-glutamyl transpeptidase activity, sodium hexose symport activity, alkaline phosphatase activity, and villin protein. This was paralleled by an increase in villin protein manifested at the single cell level. Chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited expression of proximal tubule-specific properties at the levels of enzyme activity, protein content, and mRNA content. Inhibition occurred in all cells of the population. TPA treatment induced a decrease in total protein kinase C (PKC)-alpha protein content and a change in subcellular localization from predominantly soluble to predominantly particulate. PKC-epsilon protein content was unchanged by TPA treatment. PKC-epsilon was localized in both soluble and particulate fractions of control cells but was localized predominantly in particulate fractions after TPA treatment. PKC-delta was barely detectable in control cells, but content was markedly increased by TPA. These results suggest that TPA-induced inhibition of expression of proximal tubule-specific properties is mediated through modulation of content and/or subcellular localization of one or more PKC isozymes, likely PKC-alpha.

Differential Regulation of a Multipromoter Gene.: SELECTIVE 12-O-TETRADECANOYLPHORBOL-13-ACETATE INDUCTION OF A SINGLE TRANSCRIPTION START SITE IN THE HMG-I/Y GENE

The human HMG-I/Y gene, encoding the non-histone "high mobility group" proteins HMG-I and HMG-Y, is transcriptionally activated in human K562 erythroleukemia cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induces differentiation of K562 cells within 2-4 days after treatment. In this report, we show that transcriptional activation of the HMG-I/Y gene is dependent on protein synthesis and is an early event (2 h after induction) in the TPA-mediated differentiation process. Of the four functional transcription start sites present in the gene, only one (start site 2) is preferentially induced upon TPA treatment. This is the first report, to our knowledge, of the preferential utilization of a specific transcription start site in response to a particular stimulus in a gene that contains multiple promoters. This indicates that each start site in the gene has the potential to be independently regulated instead of being coordinately controlled as shown in a number of other genes. In addition, sequences upstream of the inducible start site, which contains a TPA-responsive element, mediates TPA inducibility through AP1 (or an AP1-like) transcription factor. The HMG-I/Y proteins function as key regulators of gene expression and play a significant role in chromatin structural changes as well. The cloning and sequence analyses previously reported indicated the structure of the HMG-I/Y gene to be highly complex and predicted its expression to be tightly regulated. The results presented here confirm and extend these earlier findings.

High resolution analysis of cell cycle‐correlated vimentin expression in asynchronously grown, TPA‐treated MPC‐11 cells by the novel flow cytometric multiparameter BrdU‐hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication.

Differences in the regulation of phosphatidylinositol‐specific phospholipase C in normal and neoplastic keratinocytes

The induction of epidermal differentiation by Ca2+ in vitro is associated with enhanced activity of phosphatidylinositol-specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c-Ha-ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v-Ha-ras gene (v-Ha-ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC-gamma 1 (145 kDa) is the major isoform detected by immunoblotting of extracts from control, v-Ha-ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca2+ medium. The amount of PLC-gamma 1 protein was higher in neoplastic cell lines than in normal and v-Ha-ras keratinocytes that had similar PLC-gamma 1 protein levels. Thus, higher PLC-gamma 1 protein levels cannot account for the elevated constitutive activity PLC in v-Ha-ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC-gamma 1 protein increased in all cell types, and PLC-delta 1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased. PLC-beta 1 was not detected at any Ca2+ concentration. PLC-gamma 1 and PLC-delta 1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC-gamma 1 and PLC-delta 1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca(2+)-stimulated and basal InsP formation in neoplastic cells lines and v-Ha-ras keratinocytes. In both normal and v-Ha-ras keratinocytes labeled with [32P]orthophosphate, antibodies against PLC-gamma 1 immunoprecipitated a complex of 32P-labeled proteins. The relative labeling of the PLC-gamma 1 band was greater in normal than in v-Ha-ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC-gamma 1 in v-Ha-ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes.

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.