[Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage]. / Relations entre induction de p53, arrêt du cycle cellulaire et survie de fibroblastes humains normaux après agressions génotoxiques.

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.

Unfaithfulness and promiscuity of a mutant androgen receptor in a hormone-refractory prostate cancer.

Missense mutations in the androgen receptor (AR) contribute to the failure of hormonal therapy for prostate cancer (PCa), but the underlying molecular bases remain uncharacterized. Here, we describe a new AR variant found in a hormone-refractory metastatic PCa, in which threonine 575 in the DNA binding domain, and threonine 877 in the ligand-binding domain, were both replaced by an alanine. Using gene reporter assays, we demonstrate that the T575A mutation weakened transcriptional activity from promoters containing AR-specific responsive elements, while activity from promoters with AR-non-specific elements was enhanced. Data from gel shift experiments revealed a preferential binding of the T575A mutant to AR-non-specific motifs. We demonstrate that the two mutations T575A and T877A cooperate to confer new functional properties on the AR, and that the mutant AR functions simultaneously as a promiscuous AR due to the T877A mutation, and an unfaithful AR due to the T575A mutation.

Alternative splicing of the dopamine D2 receptor directs specificity of coupling to G-proteins.

Two isoforms of the dopamine D2 receptor have been characterized, D2L (long) and D2S (short), generated by alternative splicing from the same gene. They differ by an in-frame insert of 29 amino acids specific to D2L within the putative third intracytoplasmic loop of the receptor. We have previously demonstrated (Montmayeur, J.-P., Guiramand, J., and Borelli, E. (1993) Mol. Endocrinol. 7, 161-170) that D2S and D2L, although presenting very similar pharmacological profiles, couple differently to the alpha-subunit of guanine nucleotide-binding regulatory proteins (G-proteins). In particular, D2L, but not D2S, requires the presence of the alpha-subunit of the inhibitory G-protein (G alpha i2) to elicit greater inhibition of adenylyl cyclase activity. The insert present in D2L must therefore confer the specificity of interaction with G alpha i2. Thus, we introduced substitution mutations within the D2L insert. These mutant receptors were expressed in JEG3 cells, a G alpha i2-deficient cell line, scoring for those presenting an increased inhibition of adenylyl cyclase by dopamine. Our analysis identified two mutants, S259/262A and D249V, with these properties. These results clearly show that the insert present in D2L plays a critical role in the selectivity for the G-proteins interacting with the receptor.

Conflicting effects of caffeine on apoptosis and clonogenic survival of human K1 thyroid carcinoma cell lines with different p53 status after exposure to cisplatin or UVc irradiation.

Caffeine has been widely described as a chemo/radiosensitizing agent, presumably by inhibiting DNA repair, and affecting preferentially cells with an altered p53 status. We evaluated the effects of caffeine using isogenic and isophenotypic K1 cells derived from a papillary thyroid carcinoma and displaying either a wild type or a mutated p53 status. Apoptosis and clonogenic survival were examined after exposure of the cells to cisplatin or UVc irradiation. We find that at the most currently used concentration, 2mM, caffeine hinders cisplatin or UVc induced apoptosis in K1 cells. In addition, at this already barely achievable concentration in vivo, caffeine does not decrease their clonogenic survival. Hence in our cellular model, caffeine does not behave as a chemo- or a radiosensitizer. Although surprising, these results (1) are in agreement with the delayed G2/M block caused by caffeine that we previously observed in normal human fibroblasts and K1 cells and (2) allow us to elucidate some discrepancies concerning this molecule throughout the literature such as increase or decrease of apoptosis and clonogenic survival, activation or deactivation of molecules involved in DNA damage repair and proliferation inhibition but accelerated G2/M traverse.

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest.

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2/M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2/M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2/M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2/M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.

A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage.

BACKGROUND: Several methods have been developed for studying the kinetics of DNA repair after exposure of cells to ultraviolet (UV) light, such as conventional assays measuring unscheduled DNA synthesis (UDS). In this study, we have developed an accurate and rapid method to follow DNA gap filling during nucleotide excision repair (NER) in normal human fibroblasts (NHFs) in response to UV-induced damage. METHODS: After UVc irradiation, aphidicolin was added to the culture to hold repair patches open. This allowed an efficient incorporation of biotin-21-dUTP during an endogenous DNA repair synthesis that was detected by flow cytometry. RESULTS: We showed that the DNA gap filling after UVc irradiation in NHFs increased with time up to 10 h after irradiation and that no repair synthesis activity could be detected in XP-A fibroblasts. Furthermore, this activity was UVc dose dependent up to 20 J/m2. These results correlated well with those of the UDS assay. Interestingly, addition of aphidicolin at different time points after UVc irradiation, thus allowing endogenous repair synthesis in the absence of biotin-21-dUTP, demonstrated that the response of the NER system occurred extremely rapidly after irradiation. CONCLUSIONS: This method may be a reliable and simple alternative to other techniques measuring UDS. Practical advantages include the rapidity of the method, no need for radioactivity, and the possibility to use a second and/even a third flow marker to analyse cell cycle and heterogeneous cell populations concomitantly.

Geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells.

Geraniol and other monoterpenes found in essential oils of fruits and herbs have been suggested to represent a new class of agents for cancer chemoprevention. As a first step in clarifying the mode of action of geraniol on colon carcinogenesis, we studied its effects on the growth of a human colon cancer cell line (Caco-2). Geraniol (400 microM) caused a 70% inhibition of cell growth, with cells accumulating in the S transition phase of the cell cycle, and concomitant inhibition of DNA synthesis. No signs of cytotoxicity or apoptosis were detected. Geraniol caused a 50% decrease of ornithine decarboxylase activity, a key enzyme of polyamine biosynthesis, which is enhanced in cancer growth. This led to a 40% reduction of the intracellular pool of putrescine. Geraniol also activated the intracellular catabolism of polyamines, indicated by enhanced polyamine acetylation. These observations indicate that polyamine metabolism is presumably a target in the antiproliferative properties of geraniol.

Angiogenesis inhibitor SR 25989 upregulates thrombospondin-1 expression in human vascular endothelial cells and foreskin fibroblasts.

The aim of this work was to evaluate the effects of SR 25989, a member of the thienopyridine family devoid of antiplatelet activity but possessing anti-angiogenic properties, on the regulation of proteins involved in matrix remodeling during wound healing or tumor progression. Human endothelial cells grown in the presence of SR 25989 showed moderate increases in the production of activators (tissue plasminogen activator and urokinase) and one inhibitor (plasminogen activator inhibitor type 1) of fibrinolysis, together with a significant rise in intracellular thrombospondin-1. SR 25989 induced a similar increase in thrombospondin-1 in human foreskin fibroblasts. This over-expression of thrombospondin-1 was correlated to a decrease in cell density. A concomitant increase in the tumor suppressor gene protein p53 was observed in endothelial cells and in fibroblasts, in which the slowing down of proliferation could be related to an accumulation of cells in the S and G2/M phases of the cell cycle. Northern blot analysis revealed a temporary rise in thrombospondin-1 transcripts, followed by a decrease along with a moderate increase in p53 transcripts. Thus the anti-angiogenic properties of SR 25989 appear to result from an upregulation of thrombospondin-1 which is possibly mediated by p53. The thienopyridine SR 25989 could therefore be a good candidate for adjuvant anti-angiogenic therapy in cancer.

Caffeine and the G2/M block override: a concept resulting from a misleading cell kinetic delay, independent of functional p53.

In the literature the sensitization of DNA to radiation-induced damage by caffeine has been attributed to an override of the G2/M block. This process was supposed to involve the tumor suppressor gene p53 as it was described that p53 negative cells were more sensitive to checkpoint inhibition by caffeine than the wildtype phenotype. We have recently shown that caffeine does not cause an override of the G2/M block induced by radiation in normal human fibroblasts. We demonstrate here that this also applies to a human transformed cell line, the thyroid carcinoma K1, when submitted to gamma- rays irradiation. Within 9 hr after irradiation over 70% of the cells accumulated in the G2/M phase. This block persisted at 16 hr. In caffeine containing cultures the percentage of cells attaining the G2/M phase was reduced by over 30% at 16 hr. This was reflected in an accumulation of the cells in G1 phase and an inhibition of the S phase traverse. Cell cycle analyses from further time points combined with cell proliferation measurements confirmed these data. These results were independent of p53 status as experiments performed with variant K1 cell lines having defective p53 functions, led to similar conclusions. In addition, caffeine restored a G1 delay after irradiation in the cell lines with abrogated p53 functions. The effects of caffeine undeniably cumulate with damages induced by irradiation but probably by inhibiting DNA repair mechanisms or by intervening with purine and pyrimidine metabolisms and not by causing a G2/M block override.

Inactivation of p53 in normal human cells increases G2/M arrest and sensitivity to DNA-damaging agents.

p53 mutations are found in about 70% of human cancers. In order to evaluate the role of these mutations in response to chemotherapeutic agents, it is important to distinguish between p53 response to DNA-damaging agents in normal and in tumour cells. Here, using normal human fibroblasts (NHFs), we show that cisplatin and UV radiation induce G2/M arrest which is temporally linked to p53-protein induction. To study the contribution of p53 to this G2/M arrest, we inhibited p53 induction in NHFs using p53 anti-sense oligonucleotides. Following exposure of NHFs to UV radiation, the inhibition of p53-protein induction leads to a greater accumulation of cells in the G2/M phase, but also to a decreased fraction of cells in the G1 phase. We propose that p53 does not induce G2/M arrest directly, and that the extent of this arrest may depend on the fraction of cells that do not stop at the G1 phase following exposure to DNA-damaging agents. Furthermore, inhibition of p53-protein induction leads to increased sensitivity of NHFs to UV radiation. These results suggest that inhibition of p53 protein enhances sensitivity to DNA-damaging agents in normal human cells.

Anti-angiogenic effects of the thienopyridine SR 25989 in vitro and in vivo in a murine pulmonary metastasis model.

Neovascularisation is a key step in tumour growth and establishment of distant metastases. We have recently demonstrated that the thienopyridine SR 25989 an enantiomer of the anti-aggregant clopidogrel (Plavix) lacking anti-aggregant activity, inhibits endothelial cell proliferation in vitro by increasing the expression of endogenous thrombospondin-1, a natural potent inhibitor of angiogenesis. The anti-angiogenic effect of SR 25989 was further assessed in vitro in a quantitative assay of angiogenesis comprising a fragment of rat aorta embedded in a fibrin gel and in vivo in a pulmonary metastatic model using C57BL/6 mice inoculated in the foot pad with the highly metastatic melanoma cell line B16 F10. SR 25989 induced a dose dependent inhibition of spontaneous microvessel development in vitro reaching half maximal inhibition at around less than 50 microM and caused platelet derived growth factor induced angiogenesis to regress as a function of thienopyridine concentration. In vivo, SR 25989 did not alter significantly the growth rate of the primary tumour in the foot pad and did not inhibit development of inguinal nodes which appeared after amputation. However, the number and size of lung metastases were reduced in treated animals when examined at the time of sacrifice. In addition, the few metastases over 1 mm3 did not show any neovascularisation, as confirmed by negative von Willebrand immunostaining and in contrast to intense vascularisation seen in metastases developed by control mice. These results confirm that SR 25989 possesses potent anti-angiogenic properties and is able to inhibit metastatic dissemination and growth. The lack of effect on the primary tumour and inguinal nodes illustrates the complexity of the mechanisms involved in tumoural neo-angiogenesis and points out the possibility for distinct processes leading to neovascularisation in primary tumour as opposed to metastases.