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Listening in a noisy environment is challenging, but many previous studies have demonstrated that comprehension of speech can be substantially improved by looking at the talker's face. We recently developed a deep neural network (DNN) based system that generates movies of a talking face from speech audio and a single face image. In this study, we aimed to quantify the benefits that such a system can bring to speech comprehension, especially in noise. The target speech audio was masked with signal to noise ratios of -9, -6, -3, and 0 dB and was presented to subjects in three audio-visual (AV) stimulus conditions: (1) synthesized AV: audio with the synthesized talking face movie; (2) natural AV: audio with the original movie from the corpus; and (3) audio-only: audio with a static image of the talker. Subjects were asked to type the sentences they heard in each trial and keyword recognition was quantified for each condition. Overall, performance in the synthesized AV condition fell approximately halfway between the other two conditions, showing a marked improvement over the audio-only control but still falling short of the natural AV condition. Every subject showed some benefit from the synthetic AV stimulus. The results of this study support the idea that a DNN-based model that generates a talking face from speech audio can meaningfully enhance comprehension in noisy environments, and has the potential to be used as a visual hearing aid.

Knowledge of re-programming in cancer cells with metabolic differences from their normal counterparts has resulted in new examination of therapeutic approaches. Several studies of the role of tumor mitochondria in cancer have led to the development of non-genotoxic therapies which target mitochondrial proteins, function. The now well-established functions of mitochondria in apoptosis provide novel targets for tumor cell suicide. Mitochondria serve as a central hub for responses to cellular stress as well as injury. The alterations in cancer cells which result in protection from apoptosis can be targeted to inhibit proliferation. Because of the reprogramming of cancer cell metabolism involving increased glycolysis, it appears that blocking InsP(3)R Ca(2+) release or adaptive pathways in response to hypoxia by targeting HIF-1 or metabolic enzymes encoded by the HIF-1 gene represents a feasible therapeutic approach to cancer. A very early in vitro event found in tumor cells following resveratrol addition is an increase in intracellular Ca(2+), measurable within seconds. Ca(2+) release is also observed with non-toxic flavonoids and a goal to identify the sentinel targets of resveratrol as a model compound involved in calcium activation seems worthwhile. New findings of the relationship between autophagy and apoptosis are discussed. The contribution of reactive oxygen species (ROS) generated by mitochondria is also considered. New data as to how cyclophilins and VDAC are involved in mitochondrial hexokinase protection of factors that induce apoptosis are reviewed. In addition, chemotherapeutic approaches based on Akt-activated mTORC1 are described, and their relationship to the role of aerobic glycolysis in this protection., (Copyright © 2011 Wiley Periodicals, Inc.)

Tumor progression involves the acquisition of properties which include growth-factor independent cell proliferation, failure of inhibition by growth-inhibitory signals, ability to invade surrounding tissues, and to evade apoptosis, etc. Characterization of the profile or molecular signature of the tumor may permit the development of rational therapies that target the aberrant pathways. Rapidly growing tumor cells are usually associated with a high rates of glycolysis and in these cells, it may be advantageous to exploit this pathway which most likely is required for optimal synthetic needs. Combinatorial therapeutic agents which target the growth factor signal transduction pathways as well as apoptotic signaling pathways provide an opportunity for maximal therapeutic benefit.

TGF-beta1 induces cell cycle activation in mouse embryonic fibroblasts by down regulation of p27(Kip1) but it can also induce delay of EGF-induced cell cycle activation in these cells under similar conditions. In an attempt to determine the basis for these responses, the study of early TGF-beta1-induced signal transduction pathways in the presence and absence of EGF was undertaken. It is proposed that a likely target for the inhibition by TGF-beta1 of the early EGF-induced p42/p44 MAPK is at the c-Raf locus. The finding that the catalytic subunits of PKA are associated with Raf-1 within minutes following application of TGF-beta1 but not EGF in fibroblasts arrested in early G1 is suggestive of a role of PKA-Raf-1 interaction in TGF-beta1 induced delay of EGF-induced cell cycle kinetics. A model for TGF-beta1 induced translocation to the plasma membrane-associated Raf-1 is proposed. Reports that Rho-like GTPase activity is critical for the activation of TGF-beta1 downstream pathways raises the question as to whether Rho proteins are involved in these observed TGF-beta1-induced responses. Post-receptor signaling mechanisms for TGF-beta1 and cross-talk with PKA-mediated pathways are examined in an effort to explain the modulation by TGF-beta1 of mitogen-induced cell proliferation in mesenchymal cells., (Copyright 2003 Wiley-Liss, Inc.)

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., (Copyright 2001 Wiley-Liss, Inc.)

In mouse C3H 10T1/2 cells, we previously reported that TGF-beta1 first delays and later potentiates EGF-induced DNA synthesis corresponding to an inhibition of EGF-induced cyclin D1 expression at t = 13 h. We report here that in accord with DNA synthesis kinetics, TGF-beta1 initially suppresses EGF-induced cyclin D1 expression then later releases the inhibition. Furthermore, TGF-beta1 also first decreases and later potentiates the levels of EGF-activated MEK1/MAPK and PKB, indicating the existence of cross talk between TGF-beta 1- and EGF-activated signal transduction pathways. PD98059, the specific inhibitor of MEK1, significantly blocks EGF-induced DNA synthesis, whereas wortmannin, the PI3K inhibitor, exerts a modest inhibitory effect, which suggests that the activation of MEK1-MAPK pathway plays a major role in EGF-induced DNA synthesis and the activation of PI3K-PKB pathway plays a minor role. Upon examination of mechanisms underlying the cross talk, it was discovered that application of TGF-beta1 triggers a rapid association between Raf-1 and catalytic subunits of PKA, which are reported to be able to inactivate Raf-1 upon activation. Therefore, TGF-beta1 may activate PKA to inhibit the EGF-activated MEK1-MAPK pathway. The wortmannin-sensitive phosphorylation at the thr(389) site is necessary for activation of p70s6K, an important kinase involved in mitogen-stimulated protein synthesis. Although we found that EGF-stimulated p70s6K phosphorylates through a MAPK-dependent and a MAPK-independent (wortmannin-sensitive) pathway, TGF-beta1 failed to block EGF-triggered phosphorylation of p70s6K at thr(389) and thr(421)/ser(424) sites, implying that PKB inhibition by TGF-beta1 may result from inhibition of PDK1 activity instead of inhibition of PI3K activity. These data also suggest that TGF-beta1 may selectively perturb certain EGF-activated MAPK pools., (Copyright 2000 Wiley-Liss, Inc.)

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.

A significant portion of published literature is dedicated to describing the cloning and the characterization of proteins involved in the progression of the cell cycle, which govern cell growth both in cancer and normal ontogenesis. With this abundance of information, the cascading pathways of molecular events that occur in the cell cycle are proving to be exceedingly complicated. The purpose of this conference was to attract the leading clinical and basic science investigators in the growth control field with a final goal to determine how this current wealth of knowledge can be used to impact upon patient care and management by the design of novel adjuvant therapeutics specifically targeted at tumor cells and the identification of molecular diagnostic and/or prognostic markers in an efficient and cost effective manner.

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.

Previously, we found that stimulation of C3H 10T1/2 mouse fibroblasts with TGF-beta leads to the striking and rapid down-regulation of p27kip1 expression during G1 phase. Here, we demonstrate that TGF-beta treatment of C3H 10T1/2 cells does not alter the steady-state level of Kip1 message sufficiently to account for the observed down-regulation of p27. This demonstrates that TGF-beta-induced down regulation of p27kip1 occurs at a post-transcriptional level, consistent with a degradative mechanism of p27kip1 down-regulation. Epidermal growth factor (EGF) does not lead to the rapid down-regulation of p27 observed following treatment of cells with TGF-beta. Also in contrast with TGF-beta, EGF causes a strong upregulation of cyclin D1, while neither growth factor affects cdk4 protein levels. These results imply that in this cell type TGF-beta overcomes an inhibitory threshold to cdk activation by cyclin-dependent kinase inhibitors primarily through down-regulation of p27, while EGF overcomes this threshold predominantly through upregulation of cyclin D1 levels. This divergence in pathways may explain why TGF-beta-induced cell cycle kinetics are slower than those of EGF in these cells, and the ability of TGF-beta to delay EGF-induced cell cycle kinetics to its own, slower kinetics. In support of this hypothesis, TGF-beta prevents EGF-induced upregulation of cyclin D1 levels, while TGF-beta is still able to induce p27 down-regulation even in the presence of EGF. In contrast to the case with p27 degredation, neither TGF-beta nor EGF have an observable effect on the steady-state levels of p21 in this cell type.

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.

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.

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.

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.

We have investigated the effects of transforming growth factor alpha (TGF alpha) in C3H10T1/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGF alpha, which both utilize the EGF receptor for signal generation, are able to stimulate DNA synthesis in these cells with nearly superimposable kinetics; however, the stimulation by TGF alpha was slightly greater at nearly all time points assayed. This report is the first showing that TGF alpha, like EGF, vigorously induces c-myc and c-fos gene expression in these cells. A significant stimulation of c-myc and c-fos mRNA levels is observed with both TGF alpha and EGF; c-myc mRNA levels show an 8-fold induction with both mitogens, while c-fos inductions were on the order of 12 to 14-fold at maximum. However, the induction of c-myc mRNA by TGF alpha has slower kinetics than by EGF.

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.

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.

Intracellular calcium has been proposed to be an important mediator of signal transduction by various growth factors. We have studied the role of intracellular calcium in the mitogenic stimulation of C3H 10T1/2 mouse fibroblasts by epidermal growth factor and transforming growth factor alpha. We have found that both these peptides can cause a marked, transient increase in intracellular calcium levels. This rise occurs only in the presence of extracellular calcium. However, this calcium transient is not involved in the accumulation of c-fos and c-myc mRNAs which are elicited by these growth factors, since mRNA induction is observed to an equivalent degree in the absence or presence of extracellular calcium. These results demonstrate that although these growth factors cause an increase in intracellular calcium, the calcium second messenger system is not responsible for the induction of c-fos and c-myc mRNAs in C3H 10T1/2 fibroblasts.

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.

The introduction of either PGF2 alpha-(10(-7) M) or TPA (10(-7) M) stimulated, ouabain-sensitive 86Rb+ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF2 alpha at these concentrations stimulated the incorporation of 3H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na+/K+ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-10T1/2 cells there were parallel increases in 3H-TdR incorporation and ouabain-sensitive 86Rb+ influxes with 10(-7) M TPA, whereas PGF2 alpha stimulated a significant increase in 3H-TdR incorporation in 3T3-4 but not C3H-10T1/2 cells and only marginal increases in ouabain-sensitive 86Rb+ influx in both. Therefore, although there appears to be a close correlation between Na+/K+ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF2 alpha cannot be confirmed.

TPA stimulates cell cycle activation in both serum-deprived and density-inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5-4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced 3H-thymidine incorporation in density-inhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8-13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of 3H-TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8-13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G1 multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G1 phase of cell cycle.

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

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.

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.

Thalidomide did not inhibit dehydrogenase activity or growth of Ehrlich ascites tumor cells in agar. When mixed with Ehrlich ascites tumor cells in vitro, thalidomide increased the mitotic activity. The effect of the thalidomide was not altered by the addition of nicotinic or folic acid, or by vitamin B(1) or B(6). Oxygen uptake by the tumor cells was not affected by thalidomide.