Annie Valette

Transforming growth factor β1 is a potent survival factor for rat embryo motoneurons in culture

We have studied the effects of transforming growth factor beta 1 (TGF beta 1) on the survival of embryonic motoneurons in culture. For this purpose, E14 rat embryo motoneurons were purified to more than 90% homogeneity by cell sorting and cultured at low density on monolayers of cortex astrocytes. Subnanomolar concentrations of TGF beta 1 (40-500 pM) increased the survival of motoneurons 2-fold after 9-11 days in culture. The increase in choline acetyltransferase (ChAT) activity per culture caused by TGF beta 1 was attributable to its effects on survival. Comparable results were found with motoneurons cultured on lysed astrocytes, suggesting that the effects of the factor are not mediated by non-neuronal cells, but that motoneurons are a target for TGF beta 1.

Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12‐myristate 13‐acetate (PMA) induced upregulation of p21, not only in MCF‐7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA‐induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC‐induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin‐synchronised MCF‐7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr‐15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.

Purification and identification of multiple forms of dynorphin in human placenta.

Immunoreactive dynorphin (ir-Dyn) and opiate-like peptides (OLP) were measured in acid (HC1) extracts of human placenta by the use of an antibody to synthetic Dyn-(1-13) and of the displacement of [3H]-naloxone binding to rat brain homogenates, respectively. The placenta contained 57.6 pmoles per g of ir-Dyn and 134.4 pmoles per g of naloxone binding equivalents. After passage of the extract through cartridges of Sep Pak C18, half of the OLP was eluted with ir-Dyn at 35% acetonitrile (ACN), the rest being eluted at 60% ACN. Both fractions obtained from Sep Pak were chromatographed separately on Sephadex G-50, the OLP of the 35% ACN fraction coeluting with the ir-Dyn speak and that of the 60% ACN fraction being eluted at the same volume as synthetic beta-endorphin. Conversely, the fraction of OLP coeluting with synthetic leucine-enkephalin (Leu-Enk) in these two chromatographies was minimal. The Dyn-immunoreactive material was further purified by high pressure liquid chromatography on reverse phase micro-Bondapak C18 columns to give three distinct peaks corresponding to synthetic Dyn-(1-11), Dyn-(1-13) and Dyn-(1-12), respectively. Our results indicate that the human placenta contains several forms of ir-Dyn which account for about half of its endogenous OLP.

Increased level of p21 in human ovarian tumors is associated with increased expression of CDK2, cyclin A and PNCA

We have demonstrated over‐expression of the cyclin‐dependent kinase inhibitor p21 in various ovarian‐cancer cell lines as well as in ovarian‐tumor biopsies. This increase in p21 expression relative to that observed in normal ovarian epithelial cells is unrelated to proliferation index. In the present study, we found that p21 is functional, since the protein extracted from IGROV1 cells is still able to inhibit cdk2‐kinase activity. We then investigated how IGROV1 cells overcome the growth‐inhibitory function of p21. Immunofluorescence assays and subcellular fractionation showed that p21 is located in cytoplasm and nucleus both in normal and in tumoral cells. Compared with normal ovarian epithelial cells in culture, the increase in level of p21 in IGROV1 cells was found to be associated with increased expression of cdk2, cyclin‐A and PCNA proteins. In IGROV1 cells, p21 is associated with inactive cdk2/cyclin‐A complex, indicating that it acts as an inhibitory factor rather than an assembly factor. Over‐expression of cdk2 and of cyclin A observed in IGROV1 cells allows them to escape to p21‐inhibitory activity. The fact that cells from ovarian‐tumor biopsies exhibited a concomitant increase in p21 and in its partners cdk2 and PCNA suggest that ovarian‐tumor cells can tolerate high levels of functional p21 via over‐expression of other cell‐cycle‐regulatory proteins. Int. J. Cancer 76:891–896, 1998.© 1998 Wiley‐Liss, Inc.

Effects of TGF-β1 (transforming growth factor-β1) on the cell cycle regulation of human breast adenocarcinoma (MCF-7) cells

The antiproliferative effects of TGF‐β1 were investigated in a human breast adenocarcinoma cell line (MCF‐7). We report that TGF‐β1 inhibits proliferation through cell cycle arrest in G1. A MCF ‐7 cell subline (MCF‐7(‐)), in which the type II TGF‐β receptor is not detected, was shown to be resistant to TGF‐β1 growth inhibitory effect. Cdk2 kinase activity was inhibited in the MCF‐7 sensitive cell subline in parallel with the inhibition of cell cycle progression. In both sensitive and resistant cell lines, TGF‐β1 treatment did not affect cdk2, cdk4, cyclin E and cyclin D1 mRNA and protein levels. However, in the MCF‐7 sensitive cell subline, a time‐dependent increase in cells positive for p21WAF1/CIP1 nuclear localization was observed after TGF‐β1 treatment. These findings suggest that TGF‐β1 inhibition of MCF‐7 cell proliferation is achieved through a type II receptor‐dependent down‐regulation of Cdk2 kinase activity without modification of Cdk and cyclin expression, but correlated with an increase in p21WAF1/CIP1 nuclear accumulation.

Characterization of the antiproliferative activity of Xylopia aethiopica

BackgroundXylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells.ResultsWe report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 μg/ml against HCT116 colon cancer cells, 7.5 μg/ml and > 25 μg/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid.ConclusionThe main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.

Early gene responses associated with transforming growth factor-β1 growth inhibition and autoinduction in MCF-7 breast adenocarcinoma cells

In the human breast carcinoma cell line (MCF-7), exogenous TGF-beta 1 induces a dose-dependent inhibition of cell proliferation. In a MCF-7 cell subline [MCF-7(-)], which has an undetectable level of type II TGF-beta receptor, exogenous TGF-beta 1 does not inhibit cell proliferation but is still able to induce its own message. In both cell lines, TGF-beta 1 stimulates expression of c-jun, whereas a rapid, transient and marked increase in c-fos mRNA is only observed in the MCF-7 cells sensitive to the growth inhibitory effect of TGF-beta 1. Depletion of protein kinase C abolishes the c-fos but not the c-jun response to TGF-beta 1. Our results suggest that growth inhibition and autoinduction by TGF-beta 1 are mediated by different signalling pathways. In addition, a PKC-dependent increase in c-fos expression seems to be associated with the growth inhibitory effect of TGF-beta 1.

Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents.

CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis.1 The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery2, 3 and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis.3-5 Here, we report that in p53-/- colon carcinoma cells, a moderate increase in the CDC25B level is sufficient to impair the DNA damage checkpoint, to increase spontaneous mutagenesis, and to sensitize cells to ionising radiation and genotoxic agents. Using a tumour cell spheroid assay as an alternative to animal studies, we demonstrate that the level of CDC25B expression modulates growth inhibition and apoptotic death. Since CDC25B overexpression has been observed in a significant number of human cancers, including colon carcinoma, and is often associated with high grade tumours and poor prognosis1, our work suggests that the expression level of CDC25B might be a potential key parameter of the cellular response to cancer therapy.

Localization of Phosphorylated Forms of Bcl-2 in Mitosis : Co-Localization with Ki-67 and Nucleolin in Nuclear Structures and on Mitotic Chromosomes

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or duringmitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2phosphorylation on its function are still controversial. To better understand the role ofBcl-2 phosphorylation in mitosis, we studied the subcellular localization ofphosphorylated forms of Bcl-2. Immunofluorescence experiments performed insynchronized HeLa cells indicate for the first time that mitotic phosphorylated forms ofBcl-2 can be detected in nuclear structures in prophase cells together with nucleolin andKi-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in thesestructures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitationexperiments reveal that, in cells synchronized at the onset ofmitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase.Taken together, these data further support the idea that Bcl-2 could have a new functionat mitosis.

Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: Relationship with enhanced expression of transforming growth-factor-β1

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.