Cyclin A expression is under negative transcriptional control during the cell cycle.

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules.

Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of purified T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2 + CD28 whereas stimulation by anti CD2 or anti CD28 alone was not effective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2 + anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a specific complex with this element, whereas extracts prepared from cells treated with anti CD2 + anti CD28 failed to do so after cells entered a proliferative state.

Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb.

Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(-/-) or p107(-/-)/p130(-/-) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.

The retinoblastoma protein is essential for cyclin A repression in quiescent cells.

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(-/-) embryos and not in fibroblasts from p13O(-/-), p107(-/-) or even p130(-/-)/p107(-/-) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.

Economic evaluation of enoxaparin sodium versus heparin in unstable angina. A French sub-study of the ESSENCE trial.

OBJECTIVES: To perform an evaluation from the societal perspective of the cost of treatment with enoxaparin sodium versus unfractionated heparin (UFH) in patients with unstable angina and non-Q wave myocardial infarction in France. DESIGN: Four complementary cost-minimisation analyses based on the results of the Efficacy and Safety of Subcutaneous Enoxaparin in Non-Q wave Coronary Events (ESSENCE) international trial were conducted. We assessed differences in medical resource consumption and in duration of hospital stay in the whole study population (n = 3171) and for the French patients (n = 133). RESULTS: Results were consistent for the study group as a whole and for the French subgroup. Among patients treated with enoxaparin sodium, there was a statistically significant reduction in the use of angiography and percutaneous transluminal coronary angioplasty (whole group study: p = 0.024 and 0.006, respectively) and a trend towards shorter lengths of hospital stay. The differences in angiography and angioplasty rates led to estimated average net cost savings with enoxaparin sodium of French Francs (FF)1555 per treated patient (whole study population) and FF9993 (French subgroup) [1996 values]. The analyses based on the duration of hospital stay resulted in estimated net cost savings with enoxaparin sodium of between FF1014 per treated patient (whole study population) and FF2804 (French subgroup). CONCLUSION: Our study confirmed earlier results which show that enoxaparin sodium is cost saving in the treatment of unstable angina.

[Cyclin A: a good markers for the study of cell cycle control and tumor progression?]. / La cycline A: un bon marqueur pour l'étude du contrôle du cycle cellulaire et de la progression tumorale?

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies conducted in established cell lines and in primary human T lymphocytes, have demonstrated that the promoter of its gene is under negative transcriptional control in quiescent cells. The DNA sequences mediating this repression have been delineated through in vitro mutagenesis as well as in vivo genomic footprinting experiments. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). Using primary fibroblasts from either pRb(-/-), p107(-/-), p130(-/-) or p107(-/-)/p130(-/-) mice, we show in this work that mutation of the pRb gene has the more profound effect on cyclin A transcription. Finally, normal fibroblasts cultured in suspension fail to express cyclin A and can no longer enter S phase and proliferate, revealing thus a dependence of cyclin A expression on cell anchorage. Our work suggests the existence of at least two sets of regulators controlling cell cycle progression. On the one hand, proteins like cyclin D1, whose expression is a direct consequence of the activation of the ras signalling pathway and on the other hand, proteins like cyclin A which are secondary response effectors. As a result, growth factor stimulation leads to a transcriptional activation of the former set, while the transcription of the latter set is under the control of a repressor whose effect is alleviated after triggering the ras cascade. The status of pRb thus dictates whether cells continue their progression through the cell cycle when ras is mutated, probably by allowing the uncontrolled expression of critical genes like cyclin A.