Charles E. Wenner

Epidermal Growth Factor and tumor promoters prevent DNA fragmentation by different mechanisms

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.

Targeting mitochondria as a therapeutic target in cancer

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 InsP3R Ca2+ 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 Ca2+, measurable within seconds. Ca2+ 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. J. Cell. Physiol.

Biphasic role of TGF‐β1 in signal transduction and crosstalk

TGF‐β1 induces cell cycle activation in mouse embryonic fibroblasts by down regulation of p27Kip1 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‐β1‐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‐β1 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‐β1 but not EGF in fibroblasts arrested in early G1 is suggestive of a role of PKA‐Raf‐1 interaction in TGF‐β1 induced delay of EGF‐induced cell cycle kinetics. A model for TGF‐β1 induced translocation to the plasma membrane‐associated Raf‐1 is proposed. Reports that Rho‐like GTPase activity is critical for the activation of TGF‐β1 downstream pathways raises the question as to whether Rho proteins are involved in these observed TGF‐β1‐induced responses. Post‐receptor signaling mechanisms for TGF‐β1 and cross‐talk with PKA‐mediated pathways are examined in an effort to explain the modulation by TGF‐β1 of mitogen‐induced cell proliferation in mesenchymal cells.

Differential regulation of p27 and cyclin D1 by TGF-β and EGF in C3H 10T1/2 mouse fibroblasts

Previously, we found that stimulation of C3H 10T½ mouse fibroblasts with TGF‐β leads to the striking and rapid down‐regulation of p27Kip1 expression during G1 phase. Here, we demonstrate that TGF‐β treatment of C3H 10T½ 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‐β‐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‐β. Also in contrast with TGF‐β, 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‐β 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‐β‐induced cell cycle kinetics are slower than those of EGF in these cells, and the ability of TGF‐β to delay EGF‐induced cell cycle kinetics to its own, slower kinetics. In support of this hypothesis, TGF‐β prevents EGF‐induced upregulation of cyclin D1 levels, while TGF‐β is still able to induce p27 down‐regulation even in the presence of EGF. In contrast to the case with p27 degradation, neither TGF‐β nor EGF have an observable effect on the steady‐state levels of p21 in this cell type.

CELL CYCLE : MOLECULAR TARGETS FOR DIAGNOSIS AND THERAPY : TUMOR SUPPRESSOR GENES AND CELL CYCLE PROGRESSION IN CANCER

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. J. Cell. Biochem. 70:1–7, 1998.

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 μM), 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 PKCα and PKCϵ are expressed in C3H 10T1/2 cells, inhibitor studies suggest that PKCϵ 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.

Transforming growth factor alpha (TGFα) induction of c-fos and c-myc expression in C3H 10Tl/2 cells

Summary We have investigated the effects of transforming growth factor α (TGFα) in C3H10Tl/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGFα, 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 TGFα was slightly greater at nearly all time points assayed. This report is the first showing that TGFα, 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 TGFα 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 TGFα has slower kinetics than by EGF.

Perturbation of EGF-activated MEK1 and PKB signal pathways by TGF-?1 correlates with perturbation of EGF-induced cyclin D1 and DNA synthesis by TGF-?1 in C3H 10T1/2 cells

In mouse C3H 10T1/2 cells, we previously reported that TGF‐β1 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‐β1 initially suppresses EGF‐induced cyclin D1 expression then later releases the inhibition. Furthermore, TGF‐β1 also first decreases and later potentiates the levels of EGF‐activated MEK1/MAPK and PKB, indicating the existence of cross talk between TGF‐β 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‐β1 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‐β1 may activate PKA to inhibit the EGF‐activated MEK1‐MAPK pathway. The wortmannin‐sensitive phosphorylation at the thr389 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‐β1 failed to block EGF‐triggered phosphorylation of p70s6K at thr389 and thr421/ser424 sites, implying that PKB inhibition by TGF‐β1 may result from inhibition of PDK1 activity instead of inhibition of PI3K activity. These data also suggest that TGF‐β1 may selectively perturb certain EGF‐activated MAPK pools. J. Cell. Physiol. 185:107–116, 2000.

Cell Signaling and Cancer-Possible Targets for Therapy

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. J. Cell. Physiol. 223: 299–308, 2010.

Modulation of Redox Reactions Involved in DNA Synthesis by Oxygen and Artificial Electron Acceptors

The study of the oxygen requirements for DNA synthesis in C3H 10T1/2 mouse embryonic fibroblasts offers several advantages for the understanding of mitogen-induced cell proliferation. Firstly, cell cycle kinetics have been well defined, and these cells are capable of being staged in Go/Gl so that the role of oxygen in different phases of the cell cycle can be evaluated. Further, these cells are capable of withstanding oxygen deprivation conditions necessary for removal of trace levels of dissolved oxygen which would mask the correct assessment of needs for oxygen.