Targeting the cell cycle and the PI3K pathway: a possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells.

Corruption of the Rb and p53 pathways occurs in virtually all human cancers. This could be because it lends oncogene-bearing cells a surfeit of Cdk activity and growth, enabling them to elaborate strategies to evade tumor-suppressive mechanisms and divide inappropriately. Targeting both Cdk activities and the PI3K pathway might be therefore a potentially universal means to palliate their deficiency in cancer cells. We showed that the killing efficacy of roscovitine and 16 other purines and potentiation of roscovitine-induced apoptosis by the PI3K inhibitor, LY294002, decreased with increasing corruption of the Rb and p53 pathways. Further, we showed that purines differing by a single substitution, which exerted little lethal effect on distant cell types in rich medium, could display widely-differing cytotoxicity profiles toward the same cell types in poor medium. Thus, closely-related compounds targeting similar Cdks may interact with different targets that could compete for their interaction with therapeutically-relevant Cdk targets. In the perspective of clinical development in association with the PI3K pathway inhibitors, it might thus be advisable to select tumor cell type-specific Cdk inhibitors on the basis of their toxicity in cell-culture-based assays performed at a limiting serum concentration sufficient to suppress their interaction with undesirable crossreacting targets whose range and concentration would depend on the cell genotype.

Underlying principles of cell fate determination during G1 phase of the mammalian cell cycle.

Upon their exit from mitosis, mammalian cells enter a G(1) phase during which they acutely sense all sorts of environmental stimuli. On the basis of these signals that they first need to decipher and integrate, they decide whether to undergo division, differentiation, senescence or apoptosis. We questioned whether, despite the complexity of the G(1) regulatory network, simple organizing principles might be identified that could explain how specific input signals are converted into appropriate cell fates. For this purpose, we formulated a mathematical model of the G(1) regulatory network using a simplified description of activities linked to signal transduction, cell growth, cell division and cell death. Bifurcation analysis of the model revealed the existence of multistability between several attractor states corresponding to G(0)-arrest, G(1)-arrest, S-phase entry and apoptosis cell fates. We further unravelled interlinked feedback and feedforward loops within the G(1) regulatory network that drive the signal-dependent transition between G(0) arrest and the other cell fates. Initially, exit from G(0) and progression in early G(1) entail growth factor-dependent activation of an upstream positive feedback loop that activates the cell-growth machinery. Once ribosome synthesis is restored in G(1), a competition develops between a downstream positive feedback loop, which, upon activation, triggers S phase entry, and stress-activated pathways that promote G(1) arrest. If S phase entry prevails over G(1) arrest, cells are sensitized to apoptosis due to stress-induced activation of pro-apoptotic pathways or repression of pro-survival pathways. Thus, the choice between the four possible cell fates in the G(1) phase relies on the flexibly interlinked growth-activatory and division-activatory modules, certain components of which have antagonistic effects on pathways involved in driving apoptosis and G(1) arrest. The final outcome ultimately depends on the context-dependent coordination between the cell-growth and cell-division processes.

The flexible evolutionary anchorage-dependent Pardee's restriction point of mammalian cells: how its deregulation may lead to cancer.

Living cells oscillate between the two states of quiescence and division that stand poles apart in terms of energy requirements, macromolecular composition and structural organization and in which they fulfill dichotomous activities. Division is a highly dynamic and energy-consuming process that needs be carefully orchestrated to ensure the faithful transmission of the mother genotype to daughter cells. Quiescence is a low-energy state in which a cell may still have to struggle hard to maintain its homeostasis in the face of adversity while waiting sometimes for long periods before finding a propitious niche to reproduce. Thus, the perpetuation of single cells rests upon their ability to elaborate robust quiescent and dividing states. This led yeast and mammalian cells to evolve rigorous Start [L.H. Hartwell, J. Culotti, J. Pringle, B.J. Reid, Genetic control of the cell division cycle in yeast, Science 183 (1974) 46-51] and restriction (R) points [A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 1286-1290], respectively, that reduce deadly interferences between the two states by enforcing their temporal insulation though still enabling a rapid transition from one to the other upon an unpredictable change in their environment. The constitutive cells of multi-celled organisms are extremely sensitive in addition to the nature of their adhering support that fluctuates depending on developmental stage and tissue specificity. Metazoan evolution has entailed, therefore, the need for exceedingly flexible anchorage-dependent R points empowered to assist cells in switching between quiescence and division at various times, places and conditions in the same organism. Programmed cell death may have evolved concurrently in specific contexts unfit for the operation of a stringent R point that increase the risk of deadly interferences between the two states (as it happens notably during development). But, because of their innate flexibility, anchorage-dependent R points have also the ability to readily adjust to a changing structural context so as to give mutated cells a chance to reproduce, thereby encouraging tumor genesis. The Rb and p53 proteins, which are regulated by the two products of the Ink4a-Arf locus [C.J. Sherr, The INK4a/ARF network in tumor suppression, Nat. Rev., Mol. Cell Biol. 2 (2001) 731-737], govern separable though interconnected pathways that cooperate to restrain cyclin D- and cyclin E-dependent kinases from precipitating untimely R point transit. The expression levels of the Ink4a and Arf proteins are especially sensitive to changes in cellular shape and adhesion that entirely remodel at the time when cells shift between quiescence and division. The Arf proteins further display an extremely high translational sensitivity and can activate the p53 pathway to delay R point transit, but, only when released from the nucleolus, 'an organelle formed by the act of building a ribosome' [T. Mélèse, Z. Xue, The nucleolus: an organelle formed by the act of building a ribosome, Curr. Opin. Cell Biol. 7 (1995) 319-324]. In this way, the Ink4a/Rb and Arf/p53 pathways emerge as key regulators of anchorage-dependent R point transit in mammalian cells and their deregulation is, indeed, a rule in human cancers. Thus, by selecting the nucleolus to mitigate cell cycle control by the Arf proteins, mammalian cells succeeded in forging a highly flexible R point enabling them to match cell division with a growth rate imposed by factors controlling nucleolar assembling, such as nutrients and adhesion. It is noteworthy that nutrient control of critical size at Start in budding yeast has been shown recently to be governed by a nucleolar protein interaction network [P. Jorgensen, J.L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of pathways that couple cell growth and division in yeast, Science 297 (2002) 395-400].

Human p14(Arf): an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function.

The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16(Ink4a) which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14(Arf), a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14(Arf) and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14(Arf) predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon short-term treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14(Arf) might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.