Simone Vidal

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.

The use of a tyrosine-hydroxylase cDNA probe to study the neurotransmitter plasticity of rat sympathetic neurons in culture

We have compared quantitatively the effects of muscle-conditioned medium (CM) and elevated K+ concentration (40 mM) on the enzymatic activity of tyrosine hydroxylase (TH) and on TH-mRNA levels in primary cultures of rat sympathetic neurons. Northern blot analysis of RNA from cultured neurons with a 32P-labeled rat TH-cDNA probe was performed. The probe hybridized strongly with a single RNA species of 1.9 kb, similar in size to the TH-mRNA from PC12 pheochromocytoma cells. In agreement with earlier data both CM and a partially purified factor from CM increased choline acetyltransferase activity up to 200-fold and depressed TH activity by 2- to 7-fold in cultured sympathetic neurons. These effects were accompanied by a decrease in TH-mRNA level, which correlated with the decrease in TH activity. On the other hand, a culture medium supplemented with 40 mM KCl caused a 1.5- to 5-fold increase in TH activity, which was accompanied by an increase in TH-mRNA level of the same order of magnitude. As a working hypothesis, we suggest that CM and neuronal depolarization control the transcription of the TH gene in an antagonistic manner.

Comparison of the effects of elevated K+ ions and muscle-conditioned medium on the neurotransmitter phenotype of cultured sympathetic neurons☆

Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons. We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures. A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold. In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development. The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules. Conversely, K+ ions (30-40 mM) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold. Cultures grown with CM in high K+ medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K+ medium suggesting that CM and K+ ions had antagonistic effects on the expression of these enzymes. However, K+ ions did not affect the development of AADC in these cultures. CM suppressed in a reversible manner the development of the 16 S form of AcChE. In the presence of 40 mM K+, the rate of development of AcChE was reduced. In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed. The effect of elevated K+ ions on the percentage of 16 S AcChE was rapidly reversible. It is concluded that CM and elevated K+ ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.

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.

Induction of the vesicular monoamine transporter by elevated potassium concentration in cultures of rat sympathetic neurons

The expression of the vesicular monoamine transporter was studied in newborn rat sympathetic neurons and compared to that of the catecholamine biosynthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase. The vesicular monoamine transporter was assayed using the specific ligand [3H]dihydrotetrabenazine. In cultures grown for 10 days in the presence of 35 mM K+, tyrosine hydroxylase activity and the density of [3H]dihydrotetrabenazine binding sites were increased by a similar 2-3-fold factor, while dopamine-beta-hydroxylase activity and protein level were unchanged. Under these conditions, choline acetyltransferase activity was depressed by 90%. The induction of the vesicular monoamine transporter by high K+ was dependent upon Ca2+ entry through slow calcium channels since it was inhibited by the diphenylbutylpiperidine antagonist fluspirilene and by 20 mM Mg2+, and was enhanced by the dihydropyridine agonist, Bay K8644. The induction of the vesicular monoamine transporter by neuronal depolarization indicates the existence of a Ca2(+)-dependent mechanism of coregulation for this intrinsic component of monoaminergic synaptic vesicles and tyrosine hydroxylase. On the other hand, the apparent absence of dopamine-beta-hydroxylase induction is probably due to the continuous secretion of this intravesicular enzyme by the depolarized sympathetic neurons, an effect already observed in trans-synaptically stimulated adult sympathetic ganglion and adrenal medulla.

The role of Ca2+ channels of the L-type in neurotransmitter plasticity of cultured sympathetic neurons

We have studied the effects of Ca2+ antagonists and agonists on the development of choline acetyltransferase (ChAT), tyrosine hydroxylase (TOH) and acetylcholinesterase (AChE) in cultures of rat sympathetic neurons maintained for 6-9 days in low K+ (5 mM) or high K+ (35 mM) medium. Previous experiments have shown that high K+ medium increases TOH activity and TOH-mRNA level up to 3.5-fold and depresses the development of AChE, in particular of its asymmetric A12 form. Moreover, high K+ medium inhibits ChAT induction by 90% in muscle-conditioned medium (Raynaud et al., Dev. Biol., 119 (1987) 305-312; 121 (1987) 548-558). None of the Ca2+ antagonists tested affected the development of ChAT, TOH or AChE in low K+ medium. In high K+ medium, nitrendipine (3 microM) or fluspirilene (1 microM) fully restored ChAT induction by conditioned medium to the level observed in low K+ medium. Other drugs (1 microM) gave partial reversion: flunarizine greater than (+)-PN 200-110 greater than (-)-D-888 greater than cinnarizine = lidoflazine. On the other hand, ChAT induction was not restored by a calmodulin inhibitor, calmidazolium (1 microM). Fluspirilene, PN 200-110, and nitrendipine also totally abolished TOH induction by high K+ medium; fluspirilene (1 microM) suppressed the inhibitory effect of high K+ medium on AChE development and restored the development of A12 AChE. Conditioned medium also depresses AChE and blocks the development of A12 AChE (Swerts et al., Dev. Biol., 103 (1984) 230-234), but these effects were insensitive to fluspirilene. The Ca2+ agonist Bay K 8644 (1 microM) potentiated the effects of elevated K+ on both ChAT and TOH. The data suggest that the effects of long-term depolarization on ChAT, TOH and AChE are mediated by Ca2+ entry specifically through voltage-sensitive channels of the L-type. Our results on cultured sympathetic neurons raise the possibility that Ca2+ antagonists, which are widely used clinically, may affect the expression of neurotransmitter phenotypic traits in vivo and interfere with trans-synaptic induction of enzymes.

REGULATION OF p21WAF1/CIP1 EXPRESSION IN HUMAN OVARIAN CARCINOMA

BODART Jean-Francois’, FLAMENT..&?phanc’. BROWAEYS Edith’. BERTOUT ~arcl, ROUSSEAU Arlettel, GANNON Julian2 et VILAIN Jean-Pierrel. 1 Centre de Biologie Cellulaire, Unit6 de Dynamique des cellules embryonnaires et cancereuses, Laboratoire de Biologie du Developpcment. EA DRED 1033, UniversitC de Lille 1, SN3, F-59655 Villeneuve d’Ascq cedex, France. 2 Imperial Cancer Research Fund Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD. U.K.

Studies of the Neurotransmitter Plasticity of Cultured Rat Sympathetic Neurons at the Molecular Level

Cultures of sympathetic neurons from new-born rats constitute an attractive model to study the triggering and modulation of the expression of neurotransmitter phenotypic traits during neuronal differentiation. These neurons can express a variety of neurotransmitters and neuropeptides, and experiments performed in vivo or in cultures have led to insights on the molecular mechanisms of this phenotypic plasticity. In particular, several extracellular cues have been identified, which modify the expression of cholinergic and catecholaminergic characters in these cultured neurons (for a review, see Patterson, 1978): conditioned medium (CM) by certain non-neuronal cells induces the biosynthesis of acetylcholine in such cultures, and depress that of catecholamines (Patterson and Chun, 1977). On the other hand, neuronal depolarization fosters the development of noradrenergic characteristics and depresses acetylcholine (ACh) biosynthesis (Walicke et al., 1977; Walicke and Patterson, 1981). In addition, neuronal depolarization inhibits the development of substance P in rat sympathetic neurons, both in vivo and in culture (Kessler et al., 1981; Adler and Black, 1984).