Diane Crighton

DRAM, a p53-induced modulator of autophagy, is critical for apoptosis

Inactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.

LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.

Leading cells require LIMK for matrix degradation and invadopodia formation during collective cell migration.

DRAM Links Autophagy to p53 and Programmed Cell Death

It is clear that changes in autophagy and autophagy regulators occur during tumor development and that this can have profound effects in certain tumor settings. The fact that p53, a key tumor suppressor mutated in approximately 50% of human cancers, has now also been shown to induce autophagy, has placed autophagy center stage in the minds of those interested in the development and treatment of malignant disease. p53 is a transcription factor that responds to cellular stress and prevents the propagation of cells which may otherwise form a tumor. The recent discovery, therefore, of DRAM (damage-regulated autophagy modulator) as a new p53 target which modulates autophagy is a major step forward in understanding how p53 controls autophagy and how this relates to tumor suppression. DRAM is a lysosomal protein that is not only critical for the ability of p53 to induce autophagy, but also for p53’s ability to induce programmed cell death – a facet of p53 considered central to its tumor suppressive effects. The fact that DRAM is also inactivated in certain cancers underscores its importance and highlights the possibility that autophagy may have a more profound role in cancer than was first believed. Addendum to: DRAM : A p53-Induced Modulator of Autophagy is Critical for Apoptosis D. Crighton, S. Wilkinson, J. O’Prey, N. Syed, P. Smith, P.R. Harrison, M. Gasco, O. Garrone, T. Crook and K.M. Ryan Cell 2006; 126:121-34

On the scattering of aerodynamic noise

According to the Lighthill acoustic analogy, the sound induced by a region of turbulence is the same as that due to an equivalent distribution of quadrupole sources within the fluid. It is known that the presence of scattering bodies situated near such multipoles can convert some of their intense near field energy into the form of sound waves whose amplitude is far greater than that of the incident field. Calculations are here presented to determine the extent of this conversion, for hard and soft bodies of various shapes, making use of the reciprocal theorem to recast the problem into one of finding the field, near the obstacle, induced by an incident plane wave. If the obstacle is small compared with a wavelength, then its presence is equivalent to an additional dipole (or source) whose greater efficiency as a sound radiator implies that the familiar intensity law I ∝ U 8 , for far field intensity I against typical turbulence velocity U for an unbounded flow, is replaced by I ∝ U 6 (or I ∝ U 4 ) for a hard (or soft) body. For the situation where the scatterer is large compared with wavelength, the prototype problem of a wedge of exterior angle ( p / q )π is shown to yield an intensity law I ∝ U 4+2 q / p for both hard and soft surfaces. This result is shown to hold for the more general ‘wedge-like’ surfaces, whose dimensions are large scale and whose edges may be smoothed out on a small scale, compared with wavelength. The method used involves the matching of an incompressible flow, on the fine scales typical of the edge geometry, to an outer flow determined by the large scale features of the surface. Favourable comparisons are made with previous results pertaining to the two-dimensional semi-infinite duct and to the half-plate of finite thickness.

A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

Scattering of aerodynamic noise by a semi-infinite compliant plate

The acoustic scattering properties of a semi-infinite compliant plate immersed in turbulent flow are considered in the context of Lighthills theory of aerodynamic noise. The turbulent eddies are replaced by a volume distribution of quadrupoles, and the reciprocal theorem used to transform the quadrupole scattering problem into one of the diffraction of a plane acoustic wave. This problem is solved by the Wiener–Hopf technique for the case when elastic forces in the plate are negligible, so that a local impedance condition relates the plate velocity to the pressure difference across the plate. Strong scattering of the near-field into propagating sound occurs when certain types of quadrupole lie sufficiently close to the plate edge, and we derive explicit expressions for the scattered fields in various cases. When fluid loading effects are small, and the plate relatively rigid, the results of Ffowcs Williams & Hall (1970) are recovered, in particular the U 5 law for radiated intensity. A quite different behaviour is found in the case of high fluid loading, when the plate appears to be relatively limp. The radiated intensity then increases with flow velocity U according to a U 6 law. In aeronautical situations, surface compliance is negligible in its effect on the scattering process, and the U 5 law must then apply provided the surface is sufficiently large. On the other hand, the effect of appreciable surface compliance is to greatly inhibit the near-field scattering from the surface edge. This weaker scattering is likely to be observed in underwater applications, where fluid loading effects are generally so high as to render unattainable the condition for a plate to be effectively rigid.

Sound Generation by Turbulent Two-Phase Flow,

Abstract : Sound generation by turbulent two-phase flow is considered by the methods of Lighthills theory of aerodynamic noise. An inhomogeneous wave equation is derived, in which the effects of one phase on the other are represented by monopole, dipole and quadrupole distributions. The resulting power outputs are obtained for the case of a distribution of small air bubbles in water. The monopole radiation resulting from volumetric response of the bubbles to the turbulent pressure field overwhelms that from the quadrupoles equivalent to the turbulent flow, the increase in acoustic power output being about 70 dB for a volume concentration of 10%. The monopole radiation occurs through the forced response of the bubbles at the turbulence frequency; resonant response is shown to be impossible when the excitation is due to turbulence alone. Surface radiation arises from the edge of a cloud of bubbles. This radiation is important when the region containing bubbles is in the form of a sheet with thickness smaller than the length scale of the turbulent motion. Dipole radiation is also considered, and found to be negligible whenever monopole sources are present. In the case of a dusty gas, only dipole and quadrupole sources are present, and here it is shown that the dipole radiation will dominate that from the turbulence quadrupoles when the Mach number of the flow is less than the mass concentration of dust. (Author)

p53-mediated transcriptional regulation and activation of the actin cytoskeleton regulatory RhoC to LIMK2 signaling pathway promotes cell survival.

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents. Although RhoC and LIMK2 have well-established roles in actin cytoskeleton regulation, our results indicate that activation of LIMK2 also has a pro-survival function following DNA damage. LIMK inhibition by siRNA-mediated knockdown or selective pharmacological blockade sensitized cells to radio- or chemotherapy, such that treatments that were sub-lethal when administered singly resulted in cell death when combined with LIMK inhibition. Our findings suggest that combining LIMK inhibitors with genotoxic therapies could be more efficacious than single-agent administration, and highlight a novel connection between actin cytoskeleton regulators and DNA damage-induced cell survival mechanisms.

The excess noise field of subsonic jets

The sound field generated by the interaction of spatial instabilities on the shear layer shed from a duct with the nozzle lip is studied. It is shown that the intensity varies with direction θ from the exhaust and with the subsonic exhaust speed U according to I ∼ U 6 (1 − cosθ) 2 and I ∼ U 6 sin 2 θ for the axisymmetric and first azimuthal (sinuous) modes respectively. The first of these results is interpreted in terms of monopole and dipole sources at the exit plane, representing the acoustic effect of fluctuating mass flow and axial thrust across the exit plane, and the second in terms of a transverse dipole at the exit plane, corresponding to fluctuations in cross-stream thrust. A correlated thrust fluctuation of 1% is shown to overwhelm the jet mixing noise in the forward arc, θ > 90° while the acoustic efficiency of the interaction process is never less than 10 −6 M 3 even under the cleanest possible exit conditions. Forward flight of the duct a t Mach number M α is shown to increase the forward-arc intensity by the factor (1 + M α cos θ) −4 . It is suggested that much of the discrepancy between the noise fields of real engines and the predictions of Lighthills theory of jet mixing noise – the so-called ‘excess noise’ problem – can be explained in terms of this interaction mechanism.

p53-mediated induction of Noxa and p53AIP1 requires NFκB

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFκB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFκB. The mechanistic basis behind these observations, however, is currently incomplete. We report here further insights into this interplay using a system where blockade of NFκB inhibits cell death from p53, but at the same time sensitizes cells to death by TNFα. We found in agreement with a recent report showing that NFκB is required for the efficient activation of the BH3-only protein Noxa by the p53 family member p73, that p53’s ability to induce Noxa is also impeded by inhibition of NFκB. In contrast to the regulation by p73, however, blockade of NFκB downstream of p53 decreases Noxa protein levels without effects on Noxa mRNA. Our further analysis of the effects of NFκB inhibition on p53 target gene expression revealed that while most target genes analysed where unaffected by blockade of NFκB, the p53-mediated induction of the pro-apoptotic gene p53AIP1 was significantly dependent on NFκB. These studies therefore add further insight into the complex relationship of p53 and NFκB and since both Noxa and p53AIP1 have been shown to be important components of p53-mediated cell death responses, these findings may also indicate critical points where NFκB plays a pro-apoptotic role downstream of p53.