research reports

University of York

2006/7

Dr. James M. Allan, Department of Biology
The Role of DNA Mismatch Repair in Therapy Induced Gastrointestinal Cancer
Personnel Employed: Mrs. Sheila Taylor, Department of Biology, University of York.

Project Report: Working with Dr. Lois Travis (National Cancer Institute, United States of America), Professor Matthew Seymour, Dr. Susan Richman (University of Leeds) and Dr. Daniel Scott (Harrogate NHS Trust) we have embarked on a study to investigate the role of DNA mismatch repair in the aetiology of therapy-induced gastrointestinal cancer.

The occurence of independent second cancers has emerged as a serious long-term side-effect of chemotherapy and radiotherapy for a previous primary cancer. Understanding how and why such cancers develop could allow for preventive or surveillance measures to be implemented. Evidence suggests that defective DNA mismatch repair is a common feature of therapy-induced myeloid leukaemia, and abrogation of this pathway may also be a feature of other therapy-induced cancers.

We have demonstrated that loss of DNA mismatch repair is a relatively common feature of therapy-related stomach cancer (occuring in approximately 25% of cases), and that this is associated with concomitant genomic instability in some cases. Moreover, our data suggest that some patients may be genetically predisposed to developing multiple primary cancers with somatically acquired DNA mismatch repair defects. In order to test this hypothesis we have embarked on a genome wide search for allelic gene variants that predict risk of developing therapy-related gastrointestinal cancer. Once identified, such variants will allow for patients at high risk of therapy-related disease to be identified.

Initiation of DNA replication is a major control point in the mammalian cell cycle and the point of action of many oncogenes and tumour suppressor genes. It is also a prime target for intervention in cancer therapy. The initiation process involves assembly of replication proteins inside the nucleus during G1 phase, followed by their activation to begin DNA synthesis. This is regulated by cyclin-dependent protein kinase 2 (cdk2) and by proteins that impinge on cdk2 activity, including the cdk inhibitor p21cip1. We identified a novel contributor to this process, Cip1-interacting zinc finger protein 1 (Ciz1) and showed that it plays a positive role in DNA replication. Using p21 null cells we also demonstrated that that this occurs independently of interaction with p21cip1, suggesting that its role in initiation may be modulated by p21 rather than vice versa. Dissection of the Ciz1 protein (in collaboration with J. Ainscough, University of Leeds) revealed that Ciz1 activity is encoded by N-terminal sequences and that C-terminal domains link Ciz1 to the sub-nuclear framework (nuclear matrix) that organises replication in time and space. Furthermore, matrix-associated Ciz1 foci strikingly co-localise with sites of newly synthesized DNA in early S phase nuclei, indicating that Ciz1 is present in the DNA replication factories responsible for duplicating 'active' chromatin. The data suggest that Ciz1 plays a key role in initiation of DNA replication in mammalian cells, probably as a mediator between the DNA replication machinery and the nuclear matrix. This could contribute to the formation or function of DNA replication factories in mammalian cells.

Cip1-interacting zinc finger protein 1(Ciz1) stimulates initiation of mammalian DNA replication and is normally tethered to the nuclear matrix within DNA replication factories. We are studying an alternatively spliced human CIZ1 variant, lacking exon 4 (DE4) that is commonly detected in cancer-derived human expressed sequence tags. We find that the DE4 variant is mis-expressed as a consequence of recurring mutations in intronic splicing regulators in Ewings Tumour (ET) cell lines. Furthermore, (in collaboration with J. Ainscough and N. Copleland) we have used functional assays that reconstitute initiation of DNA replication in vitro to show that DE4 protein retains its DNA replication activity, but fails to localize to sub-nuclear foci appropriately. Co-expression of DE4 with Ciz1 prevents Ciz1 from localizing appropriately, having a dominant negative effect on foci formation. The data show that conditional exclusion of exon 4 influences the spatial distribution of Ciz1 within the nucleus, and possibly therefore the spatial organization of DNA replication. Thus, Ciz1 mis-splicing in cancer cells could influence the formation of cancer cell lineages by disrupting patterns of DNA replication and their co-ordination with gene expression. Ongoing work will systematically survey Ciz1 splice-variant expression in a range of human cancers and during normal development, using Ciz1 specific exon junction microarrays.

Personnel Employed: Dr AT Collins, Dr S Bryce, Mr P Berry, Mrs K Hyde, Ms C Brinham, Prof N J Maitland

Collaborators:
Mr M Stower, York Hospital NHS Trust
Prof F Hamdy, Prof N Brown, Dr K Linton- University of Sheffield
Dr D Hudson, Prof C Cooper- ICR Sutton, Surrey
Prof J Southgate, Dr V Knauper- Department of Biology, University of York
Mr N W Clark, Dr M Brown- CRC Paterson Institute for Cancer Research and Christie Hospital NHS Trust, Manchester
Dr G Van Leenders, Dr G Jenster- Erasmus University, Rotterdam
Dr G van der Pluijm- Leiden University Medical Center
Dr M Cecchini- BERN
Dr J Shalken- RUNMC
Dr M Campbell - University of Birmingham
Prof H Leung- Beatson Institute, Glasgow

Our laboratory reported in 2005 that a small population of cells within a prostate cancer (less than 0.1%) with a phenotype of a2b1 integrin high /CD133+ /CD44+ could be considered as prostate cancer stem cells (CSC's), ie the tumour initiating fraction, within any prostate tumour. The existence of this population of cells provides a molecular explanation for the difficulties experienced in treating prostate cancer by conventional means such as radiotherapy, chemotherapy and even immunotherapy, directed against differentiated cell antigens. The phenotype of the CSC's is a primitive basal type which differentiates in contact with a combination of appropriate basement membrane and stromal cells to form a more luminal cell expressing androgen receptor and prostate specific antigen.

Building on our expertise in the culture of these cells we have now shown that small numbers of cells are capable of inducing tumour growth when implanted orthotopically into the prostate of immunodeficient mice. These xenografts recapitulate the differentiation programme of the prostate, ie expressing androgen receptor and in some cases have been shown to metastasise from the prostate to other sites within the body of the mice.

The latency period of these tumours is relative long which imposes certain constraints on the strain of mice to be used. Current experiments will allow us to monitor tumour induction and the earlier stages of prostate cancer spread while providing a model for prostate carcinogenesis in which new cancer related therapeutics can be effectively tested before translation to human patients.

Pesonnel Employed: Dr A T Collins, Dr S D Bryce, Prof N J Maitland

Collaborators:
Mr M Stower- York Hospital NHS Trust
Dr R Birnie- Pro-Cure Therapeutics Ltd, York
Dr J Lewis- YCR Cancer Research Unit
Dr Y-J Lu -St Bartholomews Hospital, London
Prof C Cooper, Dr J Clark- Institute of Cancer Research, London

The existence of a small fraction of tumour initiating cells within a prostate cancer poses the question as to their origin and nature. Their phenotype suggests that they have been derived from the normal tissue stem cell in the prostate, but have gained the ability to survive without the normal constraints present in the normal gland.

To define the phenotype of these cells, we have fractionated primary cultures derived from a number of human prostate cancers and non-malignant tissues, and have analysed gene expression using Affymetrix plus 2 gene arrays. The output from these studies allows a comparison between the putative stem cell compartment from normal and cancerous cells and, furthermore, a comparison between the amplifying basal cells and the stem cell compartment from both normal and tumour cells. When the data is subjected to a hierarchical clustering analysis, RNA from tumour cells segregates as a distinct group from that of normal cells. However, only in tumours containing a Gleason pattern 4 (for which the clinical prognosis is poor and indicative of early metastatic spread) is the distinction clear. When Gleason pattern 3, which has a morphological appearance much closer to normal, is included in the analysis, no such segregation is possible. The gene sets from the microarray analysis have been analysed for statistical significance and a distinctive gene signature, comprising upregulated genes has been derived. These genes are indicative of both an invasive phenotype, which is in keeping with our previously published results, and also has emphasised and strengthened the connection between prostate cancer induction and the presence/induction of tissue inflammation.

In addition to the phenotypic examination of these cells we are currently analysing the DNA from purified cancer stem cells for the presence of diagnostic and potentially functional gene mutations (now partly funded by an award from the American Department of Defense).

Personnel Employed: Dr F Frame, Ms S Hagar, Dr J Burns, Dr A T Collins, Prof N J Maitland

Collaborators:
Dr G van der Pluijm- Leiden University Medical Center
Dr M Coles- University of York

A key component of the cancer stem cell hypothesis is the ability to regenerate tumours from a single cell which must be tested both in vitro and in vivo. This project seeks to introduce a permanent genetic label into the cancer stem cell population using lentiviruses expressing a marker gene, such as a green or red fluorescent protein, to permanently mark the cancer stem cell. Using the ubiquitous cytomegalovirus promoter this primary aim has been readily achieved in vitro, and a self-renewing population of prostate epithelial cells has been shown to express a red fluorescent marker (ds red) from an integrated lentiviral genome.

However, the long term stability of genes expressed from the cytomegalovirus promoter, both in vitro and in vivo, is not predictable, and other promoters have, therefore, been cloned into the basic lentivirus genome using the commercial 'Gateway' system.

We have now generated lentiviruses which express fluorescent markers from the previously identified promoters of genes which are overexpressed in the stem cell compartment, the amplifying compartment and the luminal compartment. Stem cells successfully transduced with a lentivirus have been selected with a drug resistance marker and have been put into in vitro and in vivo (xenograft) experiments, which will allow us to monitor the differentiation control of the marker genes.

Expression of the fluorescent markers also allows us to retrieve marked stem cells using flow cytometry and the introduction of the luciferase gene permits deep tissue imaging with the Xenogen IVIS whole body scanner and, similarly for fluorescent markers, with a Lighttools whole mouse illuminator.

There is now increasing evidence that stem cells are targeted in carcinogenesis. We have embarked upon a systematic study to determine the molecular basis of stem cell fate decisions in the prostate. We knew, from published data, that the extrinsic factors influencing prostate growth and differentiation are regulated by androgens and are secreted by the stromal cells in the prostate.

We firstly enriched for androgen receptor-positive (AR+) stroma by sorting cells on the basis of cell surface expression of integren a1b1, whose expression correlated well with AR expression. Using an Affymetrix gene expression array we then looked for androgen-regulated genes as potential candidates regulating stem cell fate decisions in prostate epithelia. This study was conducted in cells from patients with benign prostatic hyperplasia (n=12).

After treatment with androgen, 193 probes were found to be significantly differentially expressed after 14h, and 165 probes after 6h. We are currently validating two candidate genes from the 6h and three from the 14h time point, by RT-PCR. These genes have putative androgen-response elements suggesting androgen receptor binding ability. We have evaluated two androgen-responsive element constructs in prostate stromal cells by gene transfection to use as a reporter system for analysis of androgen stimulation and candidate gene expression analysis studies. Further validation will be carried out at the protein level and future work will determine the functional effects of these proteins on stem cell self-renewal and differentiation.

Pesonnel Employed: Dr R M Sharrard, Prof N J Maitland

Collaborators:
Dr V Knauper, Dr H Isaacs, Dr M Rumsby, Dr B Pownall- University of York
Prof D Alessi, Prof C P Downes- Unviversity of Dundee
Prof C Cooper, Dr J Clark- Institute of Cancer Research, Sutton, Surrey
Dr V Macaulay, Dr C McCarthy- University of Oxford

Aberrations in cellular signalling pathways are common within prostate cancers, but their nature is complex and they have proved an elusive target for pathway-specific therapeutics. We have recently focussed our attention on the Protein Kinase B, a key regulator of cell proliferation motility and survival, whose activation is regulated by phosphatidylinositol-3-kinase (PI3kinase). This in turn has been shown to be antagonised by PTEN whose loss in prostate cancers is one of the most significant phenotypic changes. We have examined the role of inactivating proteins for PI3kinase, such as PTEN and, most recently SHIP2, and their influence on PKB phosphorylation in a panel of human prostate-derived epithelial cells. Individual signalling pathways have been dissected by the use of specific inhibitors such as the PI3kinase inhibitor LY294002. Knocking down the expression of the SHIP2 phosphatase using optimised SiRNA markedly slowed PKB activation in response to treatment with the LY294002 drug, but only in the invasive PC3 cancer cell line.

Our results show that SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells, but not in other prostate cell lines where PTEN may share this role with further PIP3 degrading mechanisms.

We conclude that specific inhibition strategies for signalling pathways in prostate cancer cells will almost certainly require the use of multiple inhibitors to be effective and that individual prostate cancer cell lines showed different degrees of response to different combinations of inhibitors, which would result in the need for patient specific combinations to optimise the activity of potent highly specific kinase inhibitors.

Personnel Employed: Dr J E Burns, Ms H F Walker, Prof N J Maitland

Collaborators:
Dr A Antson, Prof K Wilson- York Structural Biology Laboratory, University of York
Dr I Morgan- Institute of Comparative Medicine, University of Glasgow
Dr S V Graham- Division of Virology, University of Glasgow
Dr S Allen, Dr W Zhang- School of Pharmaceutical Sciences, University of Nottingham
Dr E E Hernndez-Ramn - Medicina Genmica, Hospital General de Mxico, Mexico City, Mexico

Elucidation of the crystal structure of the transactivation domain (TAD) of the 16 E2 protein from the oncogenic human papillomavirus type 16 (HPV16) in our laboratory showed that a second dimerisation interface was located within this portion of the protein. By mutating amino acids that were predicted to disrupt TAD dimer formation without causing major structural changes we found that such mutations prevented E2-mediated transcriptional activation but had no effects on viral replication. Unlike wild type E2, the dimer interface mutants were unable to induce DNA loop formation when incubated with DNA containing E2 binding sites although DNA-binding was unaffected. Loop formation is predicted to be an important mechanism to enable distantly-bound transcription factors to be brought close to promoter sites during transcription initiation. Analytical ultracentrifugation also confirmed that dimer interface mutants remain monomeric.

We have also shown that over expression of HPV-16 E2 in human keratinocytes (their natural host) results in cell death. We generated HaCaT keratinocyte lines stably transfected with a vector allowing E2 expression under the control of the SV40 promoter and transactivation assays and RT-PCR showed low levels of E2 expression. Clones that survived selection showed characteristics of differentiated keratinocytes, including altered morphology and expression of differentiation-specific keratins 1 and 10 and filaggrin. Since the life cycle of HPV is dependent on terminal differentiation of the host cell, we suggest that one function of E2 in a natural infection may be to stimulate terminal differentiation and allow the life cycle to progress.

Personnel Employed: Ms K Tiemann, Ms S Swift, Dr J Burns, Prof N J Maitland

Collaborators:
Members of the EU Funded GIANT Program (co-ordinated by York)
Dr L Stanbridge, Ms H Evans, Ms R Nugent- YCR Cancer Research Unit, University of York
Prof C Bangma, Dr R Kraaij, Dr E Schenk-Braat- Erasmus University, The Netherlands
Dr A Antson- York Structural Biology Laboratory, University of York
Dr R Mistry,- Pro-Cure Therapeutics, York
-10 further collaborating laboratories
Members of the EU funded BACULOGENES Programme

Since there is no pre-existing immune response to baculovirus in humans and the complement-mediated response can be suppressed by the use of small molecules, we believe that these non-human viruses will form a useful addition to the anti-prostate cancer therapeutic armoury. Therefore, we have set out to re-target mammalian and insect viruses, enabling high efficiency and safer infection of prostate cancer cells.

A primary level of targeting has been obtained by the use of specific control sequences to express the therapeutic gene to generate a hybrid synthetic promoter with both high regulatable activity and prostate specificity.

As a secondary targeting strategy, we are exploring the neurotensin receptor, which is overexpressed in the membrane of prostate cancer cells, as a vector attachment target. The neurotensin ligand has been inserted successfully into the gp64 protein, which forms part of the membrane of 'humanised' baculoviruses. The modified viruses have a higher propensity to attach to, and successfully penetrate, human prostate cancer cells relative to normal cells. To further understand the molecular mechanisms behind this, we have made specific deletions in the gp64 protein to remove the heparan sulphate binding motifs which can cause unwanted binding to other cell types. We have also over-produced these proteins in a baculovirus expression system to generate milligram quantities of purified proteins. These proteins retain the ability to trimerise, and have been put into crystallisation trials. Small crystals were obtained, but diffraction patterns were not recorded at this stage. Knowledge of the three dimensional structure of retargeted gp64 should allow us to model and optimise this binding to the point where the baculovirus can be used efficiently and as a human medicine.

The mitogen-activated protein kinases (MAPKs) are part of a super family that includes the c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs) and p38 MAPK. The activation of the MAPK pathway involves a cascade of phosphorylation events initiated from MAP3Ks (MAP kinase kinase kinases) which phosphorylate and activate MAP2Ks (MAP kinase kinases) leading to the phosphorylation of MAPKs. MAPKs have largely been studied in the context of cellular stress, induced for example following exposure to UV irradiation, oxidative stress, inflammatory cytokines, and agents such as cisplatin and etoposide. Little is known about the functioning of these proteins under basal conditions. For this purpose we are employing RNAi to selectively knock-down individual genes in the absence of any applied stress. Initial studies indicate that some members of the MAPK enable constitutive viability of cancer vs non- cancer cells. This is independent of activation of the tumour suppressor p53 or a stress pathway. By combined co-silencing experiments we have mapped out these unique pro-apoptotic pathways which identify functional inter-dependency of additional genes. The discovery of genes that operate specifically in tumour cell lines will aid the development of new anti-cancer therapies which selectively kill cancer versus non-cancer cells.

RNA interference (RNAi) is a natural, highly conserved phenomenon that occurs in cells in which double stranded RNA duplexes are produced which target homologous mRNA for degradation. It has recently been discovered that RNAi can be induced experimentally in mammalian cells and without induction of a non-specific interferon response using short synthetic RNA duplexes 19-22 nucleotides in length (siRNAs). Work in our laboratory has exploited this natural phenomenon to selectively and efficiently silence expression of pathogenic viral genes and endogenous mammalian genes. Using this approach, our laboratory has identified several distinct basal apoptotic pathways that exist in colorectal cancer cells that operate under normal growth conditions (i.e. in the absence of stress) and are constitutively suppressed and also has shown that human papillomavirus E7 has anti-apoptotic function. Using siRNAs against different target mRNAs it is possible to silence expression of more than one gene simultaneously. Our current research is focusing on such co-silencing experiments in order to dissect further these apoptotic pathways and the factors involved, as well the molecular basis for the effects of HPV E7 on host cell survival.

The sirtuin deacetylase SIRT1 functions to monitor cellular nutritional status, enabling cell survival in the face of nutritional deprivation. It is now well established that SIRT1 also acts to negatively regulate the function of many transcription factors that are involved in the cellular response to exogenous stress stimuli. Prominent among these is p53, the pre-eminent tumour suppressor in mammalian cells, which functions to integrate a complex network of stress-response signalling pathways. Previously, we have demonstrated that loss of SIRT1 by RNA interference in epithelial cancer cells induces cell cycle arrest and/or apoptosis, but that non-cancer epithelial cells are refractory to loss of SIRT1. We are now extending those observations to investigate the regulation of p53 by SIRT1, under basal (normal growth conditions and in the absence of applied stress) and induced stress conditions, and comparing the results between non-cancer and cancer cells. We have determined that SIRT1 constitutively suppresses the stabilisation and activation of p53 in cancer but not in non-cancer cells, thereby indicating difference in SIRT1 function in the transition to a cancerous state. Thus, intrinsic regulation of p53 under basal conditions differs between cancer and non-cancer cells. Using RNA interference (RNAi) for specific gene silencing in conjunction with application of stress, we are determining differences in the requirements for p53 co-activators between non-cancer and cancer cells in response to induced DNA damage. Therefore we identify key differences in regulation of p53 in cancer and non-cancer cells, under both basal and stress conditions.

Human papilloma virus (HPV) is a common sexually transmitted viral infection in humans and it causes various cancers. HPV viral gene expression is tightly regulated by viral genome integration and RNA splicing. Although the knowledge of HPV mRNA structure and post-transcriptional regulation has improved there is little understanding of which mRNA is used for the expression of individual viral proteins. HPV oncoproteins E6 and E7 are expressed from three E6/E7 mRNAs. Here, we identify a fourth transcript, designated Sevix, encoded by integrated E6/E7 bicistronic DNA in cervical carcinoma cell lines, SiHa and CaSKi. For the first time we have identified the Sevix protein which is a ~8KDa fusion protein of E6 and E7. Exogenous expression of Sevix causes immortalization of primary human keratinocytes in cell culture indicating that it may play an important role in the immortalization of HPV infected cervical epithelial cells in vivo. Specific silencing of Sevix by RNA interference leads to S-phase accumulation in SiHa cells, suggesting Sevix may affect DNA replication in cervical cancer cells. Even though the Sevix is a minor transcript compared to other E6/E7 transcripts this study signifies that Sevix may have an impact on HPV carcinogenesis together with E6 and E7.

Cells with only one copy of a gene may behave abnormally, a phenomenon described as haplo-insufficiency (a half-dose = not enough). Such haplo-insufficiency has recently been documented for numerous tumour suppressor genes and is emerging as an important force driving cancer development. p53 haplo-insufficiency has been observed in vivo with human familial cancer in Li-Fraumeni Syndrome (LFS) and in mouse p53-knockout models of LFS. The increased tumorigenesis associated with loss of one p53 gene has been attributed to reduced p53-dependent stress responses. However the underlying biochemical basis for such attenuated responses in p53+/- cells remains unclear. The influence of p53 gene dosage on its tumour suppressive role in human cells is under investigation C is the full quotient of two p53 genes required for normal function or can a single p53 gene suffice? Areas under investigation in the last year include: p53 mRNA regulation, and epigenetic- and transcriptional-regulation of the human p53 gene. Also analysis of p53 splice variants, mRNA half-life, splicing efficiency of p53 transcripts, CpG methylation on the p53 gene, chromatin structure proximal to the p53 gene, characterization of the p53 gene in cell lines commonly used in the laboratory (HCT116), and use of RNA interference and ChIP analysis to dissect the transcriptional regulation of the p53 gene.

Antibody PAb240 detects a cryptic epitope in the DNA-binding region of p53 protein and as such is frequently used as a marker for the detection of mutant and denatured p53 proteins. Differentiation between wt and mutant p53 may be crucial as normal p53 is required for response to specific cancer therapies. Following Western analysis antibody PAb240 was reactive with only 2 out of 8 human epithelial colon carcinoma cell lines. We found no association between p53 mutation status and PAb240 recognition. Post-translational modifications, such as phosphorylation and acetylation are known to alter the function and stability of p53 proteins. We investigated p53 post-translational modifications in colon carcinoma cell lines studied and found an association between PAb240 reactivity and S37 phosphorylation. Following the expression of p53S37A in HCT116 p53-/- cells null for p53, reactivity with PAb240 was abrogated, whereas p53S37D proteins expressed in a similar manner were reactive with PAb240. Furthermore, mutant p53R175H proteins, known to react with antibody PAb240, were phosphorylated at S37. We also identify site-interdependent post-translational modifications since expression of p53S37A (but not p53S37D) greatly reduced phosphorylation at S15, S46, S315 and acetylation at K382 whilst increasing phosphorylation at S392.

Carlos P. Rubbi. Division of Surgery and Oncology, University of Liverpool, 5th Floor UCD Building Daulby Street, Liverpool L69 3GA
Email: c.rubbi@liv.ac.uk
Tel: 0044 (0)151 706 4148
Fax: 0044 (0)151 706 5826

Project report: The nucleolus controls p53 export and degradation

We have proposed that the nucleolus mediates constant degradation of p53, a concept that has made significant impact in p53 biology. Since nucleolar function is extremely sensitive to cellular stresses, impairment of its function determines that p53 can no longer be degraded, and a p53 response ensues by default. Therefore, it is now a fundamental question to determine if the nucleolus is capable of directly controlling p53 ubiquitination and/or export. This question has two-fold importance: it is crucial for understanding the stress response and for the development of strategies for therapeutic activation of p53.

I have addressed these questions by using heterokaryons, micronucleation and photobleaching experiments with a stably expressed p53-EGFP fusion protein, the first such stable clones to the best of much knowledge. Results demonstrate that p53 stabilisation is directly controlled by the nucleolus and indicate that p53 is exported from the nucleus through two pathways, only one of which leads to p53 degradation.

Using FRET microscopy between fluorescently labelled p53 and ubiquitin I have also managed to study the intranuclear distribution of ubiquitinated p53 (Ub-p53) and demonstrated its nucleolar association. Thus, this work demonstrates that the nucleolus is the main regulator of p53 stability, with a direct role in p53 ubiquitination and export. Furthermore, it has established new experimental reagents and techniques (stably expressed, normally regulated p53-EGFP and photobleaching and FRET assays) to study how the nucleolus exerts its control of p53 levels.