Some effects of the tumor promoter 12-0-tetradecanoylphorbol 13-acetate on cell interactions in vitro.

Studies were made of the effects of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), 4-O-methyl TPA (4-O-MeTPA), and 4 alpha phorbol 12,13-didecanoate (4 alpha PDD) on the aggregation of embryonic chick cells in gyratory shaker culture, a model system useful for the study of cell adhesion and cell interactions. TPA and, to a lesser extent, 4-O-MeTPA significantly reduced the neural retina aggregate size at concentrations as low as 10(-9) M and 10(-8) M, respectively. An inactive isomer, 4 alpha PDD, had no effect up to 10(-6) M. The reduction in aggregate size appeared related to promoter activity since dexamethasone, a steroid that inhibits tumor promotion by TPA, significantly reversed the inhibitory effect of TPA. None of the agents tested affected the sorting pattern in mixed neural retina and heart cultures. The results indicate that intercellular adhesion, as determined by extent of aggregation, is reduced in the presence of TPA. This inhibition is considered to be related to the tumor-promoting activity of TPA.

Surface properties of phorbol esters and their interaction with lipid monolayers and bilayers.

The potent tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) is surface active and was found to occupy a limiting area of 62 sq A/molecule in monolayers at the air-water interface. The interfacial tension of aqueous TPA solutions is decreased by increasing the bulk-phase TPA concentrations up to 2 x 10(-6) M,beyond which no further decreases were observed. This concentration is in agreement with the apparent solubility limit previously obtained. The apparent aqueous solubility limit of the more hydrophobic phorbol-didecanoate is 5 x 10(-8) M. Interaction of TPA with egg phosphatidylcholine monolayers at the air-water interface was shown by an increase in the surface pressure of the monolayer from 22 dynes/cm, initial film pressure, to 34 dynes/cm 90 min after introduction of TPA into the aqueous subphase. It was shown by gel filtration chromatography that a similar phorbol derivative, tritiated phorbol-didecanoate, binds to phospholipid vesicles. Differential scanning calorimetry also indicated that the addition of either TPA or an inactive stereoisomer, 4-alpha-phorbol-didecanoate, to phospholipid bilayers results in a marked reduction of the enthalphy of the minor transition of dipalmitoylphosphatidylcholine liposomes. Several fluorescence polarization probes for membrane fluidity indicate that TPA does not affect this membrane parameter. Further, the presence of TPA induces no measurable change in the cation permeability of phospholipid vesicles, the conductance of planar bilayer membranes, or the electrophoretic mobility of negatively charged liposomes. The lack of a specific effect with bilayers alone, combined with the documented physiological effects at low TPA concentrations, point to the possibility of a specific membrane component as the receptor for TPA at the plasma membrane.

Transforming growth factor beta-induced activation of cyclin E-cdk2 kinase and down-regulation of p27Kip1 in C3H 10T1/2 mouse fibroblasts.

Transforming growth factor (TGF-beta)-stimulated induction of DNA synthesis is preceded by the activation of cyclin E/cyclin-dependent kinase (cdk)2 kinase in late G1 in C3H 10T1/2 mouse fibroblasts. TGF-beta has no effect on the steady-state level of cdk4, while having only a modest inductive effect on cyclin D1 expression. TGF-beta stimulation does, however, lead to the striking down-regulation of p27Kip1 expression during G1 in a manner consistent with the timing of cyclin E-cdk2 activation. Coimmunoprecipitation analysis reveals that the amount of p27Kip1 in complexes with the cdk2 catalytic subunit is drastically reduced at the time in late G1 when cyclin E-cdk2 activity is maximal. These data indicate that cyclin E-cdk2 is inhibited by p27Kip1 in the growth-arrested state and that TGF-beta relieves this inhibition by down-regulating the steady-state level of the p27Kip1 inhibitor protein, thus reducing the level of inhibitor present in complexes with cdk2.

Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-beta.

The aim of this review is to provide insight into the molecular mechanisms by which transforming growth factor-beta (TGF-beta) modulates cell cycle progression in different cell types. Particular attention is focused on the differences between these mechanisms in cells of epithelial origin and in mesenchymally derived cells. This is important because many transformed epithelial cells lose responsiveness to the growth-inhibitory effects of TGF-beta, thus generating a more fibroblast-like phenotype. Loss of negative growth control, including a lack of response to growth-inhibitory factors, is a common feature of many tumor cells. G1 phase cyclin-dependent kinases (cdks) and their inhibitors (ckis) are central to the pathways that regulate commitment to cellular division in response to positive as well as negative growth effectors. Many checkpoints are deregulated in oncogenesis, and this is often due to alterations in cyclin-cdk complexes. The loss of R-point regulation, in particular, can allow cell growth and division to proceed autonomously of external signals. This may occur due to either the aberrant expression of positive regulators, such as the cyclins and cdks, or the loss of negative regulators, such as the ckis. Beginning with a survey of the role of the cdks in the mammalian cell cycle, the review examines how cdk activity is modulated by cyclin binding, phosphorylation, and ckis, including the Ink4 proteins and the closely related inhibitors p21Cip1 and p27Kip1. Particular attention is paid to the role of p27Kip1 and p21Cip1 in the mechanisms of TGF-beta-induced suppression or stimulation of the cell cycle and how these mechanisms contrast between epithelial cells and cells of mesenchymal origin. Other aspects of TGF-beta signal transduction are discussed, including its effects on cyclin and cdk expression in various cell types, and the downstream targets of cdks and their modulation by TGF-beta and other growth factors are also discussed. These include proteins of the retinoblastoma family, and the related modulation of the transcriptional activity of the E2F family members. Finally, the role of cell cycle regulatory proteins in oncogenesis is review in view of the findings described here.

The role of P(i) in glycolytic inhibition of calcium ion uptake by ELD ascites tumor cells.

In contrast to previous investigations at 25 degrees C, glucose was shown to support 45Ca2+ uptake at 37 degrees C in intact ELD ascites tumor cells. Intact ascites tumor cells in vitro accumulated up to 5.0 micromol of 45Ca2+ per g cells dry wt. within 20 min. In the presence of 10.0 mM glucose, intracellular P(i) levels fell from 40.0 micromol x g(-1) cells dry wt. to 20.0 micromol x g(-1) cells dry wt. in 5 min. Intracellular P(i) levels were maintained by 20.0 mM extracellular Tris-P(i). 45Ca2+ uptake was inhibited in P(i)-depleted cells, even though the metabolic rate (as measured by Q(lactate)) and energy state (as measured by ATP levels) were at acceptable levels. Evidence has been presented suggesting that previous reports of glucose inhibition of calcium uptake can be attributed to a competition for available intracellular P(i) between glycolytic processes and the mitochondrial calcium uptake mechanism.

Transforming growth factor beta 1-induced delay of cell cycle progression and its association with growth-related gene expression in mouse fibroblasts.

TGF beta-induced cell cycle progression is relatively slower than that induced by EGF or PDGF-BB. Further, TGF beta delays EGF or PDGF-induced 5-phase entry in C3H 10T1/2 mouse fibroblasts. In accordance with this delay, the induction of mRNA level of 'immediate early genes' such as c-myc, c-fos, c-jun and junB by TGF beta has slower kinetics compared with those of EGF. TGF beta induces c-sis gene, suggesting possible involvement of secondary growth stimulation by PDGF-like proteins. However, anti-PDGF-AB antibody, which was inhibitory to FDGF-BB-induced [3H]thymidine incorporation, did not block TGF beta-induced DNA synthesis. These results first demonstrate that the delay of cell cycle progression by TGF beta is closely associated with the altered regulation of growth-related gene expression in fibroblasts.

Perturbation of EGF-induced MAP kinase activation by TGF-beta 1.

TGF-beta 1 regulates both cellular growth and phenotypic plasticity important for maintaining a growth advantage and increased invasiveness in progressively malignant cells. Recent studies indicate that TGF-beta-1 stimulates the conversion of epitheliod to fibroblastoid phenotype which presumably leads to the inactivation of growth-inhibitory effects by TGF-beta 1 (Portella et al. (1998) Cell Growth and Differentiation, 9: 393-404). Therefore, the investigation of TGF-beta 1 signaling that leads to altered growth and migration may provide novel targets for the prevention of increased cell growth and invasion. Although much attention has been paid to TGF-beta 1 responses in epithelial cells, the above studies suggest that examination of signal transduction pathways in fibroblasts are important as well. Data from our laboratory are consistent with the concept that TGF-beta 1 can act as a regulatory switch in density-dependent C3H 10T1/2 fibroblasts capable of either promoting or delaying G1 traverse. The regulation of this switch is proposed to occur prior to pRb phosphorylation, namely prior to activation of cyclin-dependent kinases. The current study is concerned with the evaluation of a key cyclin (cyclin D1) which activates cdk4 and p27KIP1 which in turn inhibit cdk2 in the proliferative responses of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and their modulation by TGF-beta 1. Although the molecular events that lead to elevation of cyclin D1 are not completely understood, it appears likely that activation of p42/p44MAPK kinases is involved in its transcriptional regulation. TGF-beta 1 delayed EGF- or PDGF-induced cyclin D1 expression and blocked the induction of active p42/p44MAPK. The mechanism by which TGF-beta 1 induces a block in p42/p44MAPK activation is being examined and the possibility that TGF-beta 1 regulates phosphatase activity is being tested.

Modulation of cyclin D1 and its signaling components by the phorbol ester TPA and the tyrosine phosphatase inhibitor vanadate.

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) triggers cell-cycle progression at G1 phase in mouse embryonic fibroblast C3H 10T1/2 cells was examined. TPA treatment resulted in a temporary induction of cyclin D1 peaking at 9 h post stimulation. PD98059 (10 microM), the specific inhibitor of MAPK kinase, completely blocked TPA-stimulated cyclin D1 induction and DNA synthesis, confirming that MAPK activation plays an essential role in TPA-stimulated cell-cycle progression. Although both PKCalpha and PKCepsilon are expressed in C3H 10T1/2 cells, inhibitor studies suggest that PKCepsilon activation is required for the activation of MEK/MAPK signal transduction cascade. p70s6K, an important kinase involved in the regulation of protein synthesis and cell-cycle progression, has been reported to be activated through a PKC-dependent pathway (TPA-activatable) in addition to a PI3K-dependent pathway. Here, we demonstrate for the first time that TPA-stimulated MAPK activation is essential for the phosphorylation of several key residues involved in the activation of p70s6K, namely, thr389, thr421, and ser424. Vanadate, the tyrosine phosphatase inhibitor, triggered a sustained elevation of the level of active MAPK. However, corresponding to a rapid loss of cyclin D1 protein, vanadate treatment resulted in a significant shut out of 3H-thymidine incorporation into DNA regardless of TPA cotreatment. Vanadate treatment also led to the increase of active MEK, increased phosphorylation of p70s6K at thr389, thr421, and ser424 yet without activation of PKB. These data suggest that vanadate can selectively perturb the activation of signaling components which raises the interesting issue as to how vanadate downregulates the cyclin D1 level.

Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.

Correlation of ouabain-sensitive ion movements with cell-cycle activation.

The role of tumor-promoter-induced Na+, K+-ATPase activity in cell proliferation and the extent of coupling of Na+ and K+ movements to cell-cycle control under differing physiological states was examined. Earlier studies indicated that staging of cells in G1 by serum deprivation in the presence of phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induced a state(s) in which postconfluent C3H 10T1/2 fibroblasts were refractory to ouabain inhibition of DNA synthesis, and the present study further examines this property. Previous findings suggested that the promoter can act in either of two ways: (i) it can act by inducing an alternative pathway to S-phase independent of Na+,K+-ATPase activity, or (ii) the promoter can advance the cells in G1 to a point beyond which Na+, K+-ATPase activity is no longer required for the induction of DNA synthesis. When ouabain (0.3 mM) was added simultaneously with tumor promoters, such as dihydroteleocidin B, to cells arrested in the G1 phase, [3H]thymidine incorporation was inhibited greater than 90%. These data suggest that an alternative pathway is not likely the explanation but that tumor promoters advance cells through a dynamic state in G1, during which progression toward S-phase entry is independent of a Na+,K+-ATPase dependent regulatory step. Ouabain sensitivity kinetics measured by two independent methods indicated that the development of ouabain insensitivity is found in G1 approximately equal to 2 hr prior to S phase. This study indicates that the measured ion movements are markedly dependent on cell-cycle state and describes the criteria required to obtain reproducible responses.

Transforming growth factor-beta regulation of retinoblastoma gene product and E2F transcription factor during cell cycle progression in mouse fibroblasts.

The mechanism by which transforming growth factor beta (TGF beta) exerts growth stimulatory effects was examined in C3H/10T1/2 mouse fibroblasts by study of cell cycle regulation of the retinoblastoma gene product (p110Rb) and transcriptional regulation of the p110Rb-associated transcription factor, E2F. Northern blotting analysis shows that TGF beta and/or epidermal growth factor (EGF) stimulate by three to sixfold the level of Rb mRNA which is also reflected by the increased levels of p110Rb. p110Rb becomes phosphorylated in mid-G1 and further phosphorylated at the G1/S transition. Hyperphosphorylation of p110Rb by TGF beta can be observed when cells are in S phase. TGF beta stimulates by three to fourfold the activity of cdk2 kinase consistent with the observed phosphorylation of p110Rb and also with the possibility that the kinase is involved in phosphorylating p110Rb close to the G1/S transition. Thus, TGF beta as a growth stimulator induces, as does EGF, the phosphorylation of p110Rb during cell cycle progression. Transient transfection of E2F promoter constructs was used to analyze the effect of TGF beta on the modulation of E2F-mediated transcription. The data revealed that TGF beta can stimulate wild-type adenoviral E2 promoter activity by 12-fold. Taken together, TGF beta-induced phosphorylation of p110Rb in mouse fibroblasts appears to exert a positive regulatory function upon genes that have a pivotal role in the G1/S transition of the cell cycle.

Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts.

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, was used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryonic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Concentrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml)- or 10% serum-induced [3H]thymidine incorporation into postconfluent, quiescent cells. Further, these inhibitory effects were observed when okadaic acid was added as late as 10 hr after TGF-beta or serum stimulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these observations indicate that a phosphatase activity may be required for S-phase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [14C]leucine incorporation by 20-65%, depending upon the okadaic acid concentration. In conjunction with the effect of okadaic acid on DNA and protein synthesis, Western blot analysis indicated that okadaic acid inhibited phosphorylation of the retinoblastoma gene product and decreased its protein level, even when added 10 hr after TGF-beta or 8 hr after serum stimulation. These findings strongly suggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-transduction pathways for the stimulation of phosphorylation of the retinoblastoma protein, especially in late G1.