Inhibition of HDM2 and Activation of p53 by Ribosomal Protein L23

The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus

Potassium channel dysfunction in human neuronal models of Angelman syndrome

Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors

Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence

<p>Abstract</p> <p>Background</p> <p>Cellular senescence is a state reached by normal mammalian cells after a finite number of cell divisions and is characterized by morphological and physiological changes including terminal cell-cycle arrest. The limits on cell division imposed by senescence may play an important role in both organismal aging and in preventing tumorigenesis. Cellular senescence and organismal aging are both accompanied by increased DNA damage, seen as the formation of γ-H2AX foci (γ-foci), which may be found on uncapped telomeres or at non-telomeric sites of DNA damage. However, the relative importance of telomere- and non-telomere-associated DNA damage to inducing senescence has never been demonstrated. Here we present a new approach to determine accurately the chromosomal location of γ-foci and quantify the number of telomeric versus non-telomeric γ-foci associated with senescence in both human and mouse cells. This approach enables researchers to obtain accurate values and to avoid various possible misestimates inherent in earlier methods.</p> <p>Results</p> <p>Using combined immunofluorescence and telomere fluorescence <it>in situ </it>hybridization on metaphase chromosomes, we show that human cellular senescence is not solely determined by telomeric DNA damage. In addition, mouse cellular senescence is not solely determined by non-telomeric DNA damage. By comparing cells from different generations of telomerase-null mice with human cells, we show that cells from late generation telomerase-null mice, which have substantially short telomeres, contain mostly telomeric γ-foci. Most notably, we report that, as human and mouse cells approach senescence, all cells exhibit similar numbers of total γ-foci per cell, irrespective of chromosomal locations.</p> <p>Conclusion</p> <p>Our results suggest that the chromosome location of senescence-related γ-foci is determined by the telomere length rather than species differences <it>per se</it>. In addition, our data indicate that both telomeric and non-telomeric DNA damage responses play equivalent roles in signaling the initiation of cellular senescence and organismal aging. These data have important implications in the study of mechanisms to induce or delay cellular senescence in different species.</p

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation

Stem cells persist throughout life by self-renewing in numerous tissues including the central(1) and peripheral(2) nervous systems. This raises the issue of whether there is a conserved mechanism to effect self-renewing divisions. Deficiency in the polycomb family transcriptional repressor Bmi-1 leads to progressive postnatal growth retardation and neurological defects(3). Here we show that Bmi-1 is required for the self-renewal of stem cells in the peripheral and central nervous systems but not for their survival or differentiation. The reduced self-renewal of Bmi-1-deficient neural stem cells leads to their postnatal depletion. In the absence of Bmi-1, the cyclin-dependent kinase inhibitor gene p16(Ink4a) is upregulated in neural stem cells, reducing the rate of proliferation. p16(Ink4a) deficiency partially reverses the self-renewal defect in Bmi-1(-/-) neural stem cells. This conserved requirement for Bmi-1 to promote self-renewal and to repress p16(Ink4a) expression suggests that a common mechanism regulates the self-renewal and postnatal persistence of diverse types of stem cell. Restricted neural progenitors from the gut and forebrain proliferate normally in the absence of Bmi-1. Thus, Bmi-1 dependence distinguishes stem cell self-renewal from restricted progenitor proliferation in these tissues.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62726/1/nature02060.pd

Nutlin-3, the small-molecule inhibitor of MDM2, promotes senescence and radiosensitises laryngeal carcinoma cells harbouring wild-type p53

BACKGROUND: Primary radiotherapy (RT) is a mainstay of treatment for laryngeal squamous cell carcinoma (LSCC). Although the cure rates for early (T1) vocal cord tumours are high, RT proves ineffective in up to a third of T3 carcinomas. Moreover, RT is associated with debilitating early- and late-treatment-related toxicity, thus finding means to de-escalate therapy, while retaining/augmenting therapeutic effectiveness, is highly desirable. p53 is a key mediator of radiation responses; we therefore investigated whether Nutlin-3, a small-molecule inhibitor of MDM2 (mouse double minute 2; an essential negative regulator of p53), might radiosensitise LSCC cells. METHODS: We performed clonogenic assays to measure radiosensitivity in a panel of LSCC cell lines (for which we determined p53 mutational status) in the presence and absence of Nutlin-3. RESULTS: LSCC cells harbouring wild-type p53 were significantly radiosensitised by Nutlin-3 (P&lt;0.0001; log-rank scale), and displayed increased cell cycle arrest and significantly increased senescence (P&lt;0.001) in the absence of increased apoptosis; thus, our data suggest that senescence may mediate this increased radiosensitivity. CONCLUSION: This is the first study showing Nutlin-3 as an effective radiosensitiser in LSCC cells that retain wild-type p53. The clinical application of Nutlin-3 might improve local recurrence rates or allow treatment de-escalation in these patients

Id-1 and Id-2 are markers for metastasis and prognosis in oesophageal squamous cell carcinoma

Id protein family consists of four members namely Id-1 to Id-4. Different from other basic helix–loop–helix transcription factors, they lack the DNA binding domain. Id proteins have been shown to be dysregulated in many different cancer types and their prognostic value has also been demonstrated. Recently, Id-1 has been shown to be upregulated in oesophageal squamous cell carcinoma (ESCC). However, the prognostic implications of Id proteins in ESCC have not been reported. We examined the expression of the Id proteins in ESCC cell lines and clinical ESCC specimens and found that Id protein expressions were dysregulated in both the ESCC cell lines and specimens. By correlating the expression levels of Id proteins and the clinicopathological data of our patient cohort, we found that M1 stage tumours had significantly higher nuclear Id-1 expression (P=0.012) while high nuclear Id-1 expression could predict development of distant metastasis within 1 year of oesophagectomy (P=0.005). In addition, high levels of Id-2 expression in both cytoplasmic and nuclear regions predicted longer patient survival (P=0.041). Multivariate analysis showed that high-level expression of Id-2 in both cytoplasmic and nuclear regions and lower level of nuclear Id-1 expression were independent favourable predictors of survival in our ESCC patients. Our results suggest that Id-1 may promote distant metastasis in ESCC, and both Id-1 and Id-2 may be used for prognostication for ESCC patients

Mammary epithelial cell transformation: insights from cell culture and mouse models

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16(INK4a), p21, p27 and p57), p14(ARF), telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies

Radiosensitization of mammary carcinoma cells by telomere homolog oligonucleotide pretreatment

Introduction: Ionizing radiation (IR) is a widely used approach to cancer therapy, ranking second only to surgery in rate of utilization. Responses of cancer patients to radiotherapy depend in part on the intrinsic radiosensitivity of the tumor cells. Thus, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. Methods: Mammary tumor cells were treated by a 16-base phosphodiester-linked oligonucleotide homologous to the telomere G-rich sequence TTAGGG (T-oligo: GGTTAGGTGTAGGTTT) or a control-oligo (the partial complement, TAACCCTAACCCTAAC) followed by IR. The inhibition of tumor cell growth in vitro was assessed by cell counting and clonogenic cell survival assay. The tumorigenesis of tumor cells after various treatments was measured by tumor growth in mice. The mechanism underlying the radiosensitization by T-oligo was explored by immunofluorescent determination of phosphorylated histone H2AX ($\gamma$H2AX) foci, $\beta$-galactosidase staining, comet and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assays. The efficacy of the combined treatment was assessed in a spontaneous murine mammary tumor model. Results: Pretreatment of tumor cells with T-oligo for 24 hours in vitro enhanced both senescence and apoptosis of irradiated tumor cells and reduced clonogenic potential. Radiosensitization by T-oligo was associated with increased formation and/or delayed resolution of $\gamma$H2AX DNA damage foci and fragmented DNA. T-oligo also caused radiosensitization in two in vivo mammary tumor models. Indeed, combined T-oligo and IR-treatment in vivo led to a substantial reduction in tumor growth. Of further significance, treatment with T-oligo and IR led to synergistic inhibition of the growth of spontaneous mammary carcinomas. Despite these profound antitumor properties, T-oligo and IR caused no detectable side effects under our experimental conditions. Conclusions: Pretreatment with T-oligo sensitizes mammary tumor cells to radiation in both in vitro and in vivo settings with minimal or no normal tissue side effects