Replicational Stress Selects for p53 Mutation

p53 is a critical mediator of cellular responses to a variety of stresses. Given the frequency of p53 mutations in human malignancies and that disruption of p53 has been implicated in chemoresistance, understanding the factors that select for p53 disruption is important both for understanding tumor evolution and for designing cancer therapies. While it is widely believed that genotoxic stress selects for p53 mutations, the effects of DNA damaging agents on long-term proliferative potential are usually not affected by p53 status. Previous reports have demonstrated that despite being activated, p53 loss does not prevent cell cycle arrest and senescence in response to high levels of acute replicational stress. In contrast, we recently reported that chronic exposure of non-transformed cells to low, clinically relevant levels of replicational stress induces p53-dependent senescence-like arrest. Disruption of p53 or its target gene p21CIP1 antagonizes this arrest, leading to a long-term proliferative advantage. However, when replicational stress is associated with substantial DNA strand breaks, the ability of p53 disruption to up-regulate RAD51 dependent homologous recombination becomes important. Replicational stress is induced by many chemotherapeutic treatments and perhaps by some dietary deficiencies, and may be an important factor that selects for p53 mutations during cancer initiation and progression.     

Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets

The p53 tetramer recognizes specifically a 20-bp DNA element. Here, we examined symmetries encoded in p53 response elements (p53REs). We analyzed base inversion correlations within the half-site, as well as in the full-site palindrome. We found that p53REs are not only direct repeats of half-sites; rather, two p53 half-sites couple to form a higher order 20bp palindrome. The palindrome couplings between the half-sites are stronger for the human than for the mouse genome. The full-site palindrome and half-site palindrome are controlled by insertions between the two half-sites. The most notable feature is that the full-site palindrome with coupling between quarter-sites one and four (H14 coupling) dominates the p53REs without insertions. The most frequently observed insertion in human p53REs of 3bp enhances the half-site palindrome. The statistical frequencies of the coupling between the half-sites in the human genome correlate with grouped experimental p53 affinities with p53REs. Examination of known p53REs indicates the H14 couplings are stronger for positive regulation than for negatively regulated p53REs, with repressors having the lowest H14 couplings. We propose that the palindromic sequence couplings may encode such potential preferred multiple binding modes of the p53 tetramer to DNA.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.     

Depression of p53-independent Akt survival signals in human oral cancer cells bearing mutated p53 gene after exposure to high-LET radiation

Although mutations and deletions in the p53 tumor suppressor gene lead to resistance to low linear energy transfer (LET) radiation, high-LET radiation efficiently induces cell lethality and apoptosis regardless of the p53 gene status in cancer cells. Recently, it has been suggested that the induction of p53-independent apoptosis takes place through the activation of Caspase-9 which results in the cleavage of Caspase-3 and poly (ADP-ribose) polymerase (PARP). This study was designed to examine if high-LET radiation depresses serine/threonine protein kinase B (PKB, also known as Akt) and Akt-related proteins. Human gingival cancer cells (Ca9-22 cells) harboring a mutated p53 (mp53) gene were irradiated with 2 Gy of X-rays or Fe-ion beams. The cellular contents of Akt-related proteins participating in cell survival signaling were analyzed with Western Blotting 1, 2, 3 and 6h after irradiation. Cell cycle distributions after irradiation were assayed with flow cytometric analysis. Akt-related protein levels decreased when cells were irradiated with high-LET radiation. High-LET radiation increased G(2)/M phase arrests and suppressed the progression of the cell cycle much more efficiently when compared to low-LET radiation. These results suggest that high-LET radiation enhances apoptosis through the activation of Caspase-3 and Caspase-9, and suppresses cell growth by suppressing Akt-related signaling, even in mp53 bearing cancer cells.     

Inhibition of stress-inducible kinase pathways by tumorigenic mutant p53

More than 50% of human cancers contain p53 gene mutations and as a result accumulate altered forms of the full-length p53 protein. Although certain tumor types expressing mutant p53 protein have a poor prognostic process, the precise role of mutant p53 protein in highly malignant tumor cells is not well defined. Some p53 mutants, but not wild-type p53, are shown here to interact with Daxx, a Fas-binding protein that activates stress-inducible kinase pathways. Interaction of Daxx with p53 is highly dependent upon the specific mutation of p53. Tumorigenic mutants of p53 bind to Daxx and inhibit Daxx-dependent activation of the apoptosis signal-regulating kinase 1 stress-inducible kinases and Jun NH(2)-terminal kinase. Mutant p53 forms complexes with Daxx in cells, and consequently, mutant p53 is able to rescue cells from Daxx-dependent inhibition of proliferation. Thus, the accumulation of mutant p53 in tumor cells may contribute to tumorigenesis by inhibiting stress-inducible kinase pathways.     

p53 potentiation of tumor cell susceptibility to CTL involves Fas and mitochondrial pathways

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.