UNDERSTANDING NANOG'S ROLE IN CANCER BIOLOGY

Understanding Nanog’s Role in Cancer Biology Mark Daniel Badeaux Supervisory Professor Dean Tang, PhD The cancer stem cell model holds that tumor heterogeneity and population-level immortality are driven by a subset of cells within the tumor, termed cancer stem cells. Like embryonic or somatic stem cells, cancer stem cells are believed to possess self-renewal capacity and the ability to give rise to a multitude of varieties of daughter cell. Because of cancer’s implied connections to authentic stem cells, we screened a variety of prostate cancer cell lines and primary tumors in order to determine if any notable ‘stemness’ genes were expressed in malignant growths. We found a promising lead in Nanog, a central figure in maintaining embryonic stem cell pluripotency, and through a variety of experiments in which we diminished Nanog expression, found that it may play a significant role in prostate cancer development. We then created a transgenic mouse model in which we targeted Nanog expression to keratin 14-expressing in order to assess its potential contribution to tumorigenesis. We found a variety of developmental abnormalities and altered differentiation patterns in our model , but much to our chagrin we observed neither spontaneous tumor formation nor premalignant changes in these mice, but instead surprisingly found that high levels of Nanog expression inhibited tumor formation in a two-stage skin carcinogenesis model. We also noted a depletion of skin stem cell populations, which underlies the wound-healing defect our mice harbor as well. Gene expression analysis shows a reduction in c-Jun and Bmp5, two genes whose loss inhibits skin tumor development and reduces stem cell counts respectively. As we further explored Nanog’s activity in prostate cancer, it became apparent that the protein oftentimes was not expressed. Emboldened by the competing endogenous RNA (ceRNA) hypothesis, we identified the Nanog 3’UTR as a regulator of the tumor suppressive microRNA 128a (miR-128a), which includes known oncogenes such as Bmi1 among its authentic targets. Future work will necessarily involve discerning instances in which Nanog mRNA is the biologically relevant molecule, as well as identifying additional mRNA species which may serve solely as a molecular sink for miR-128a.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Metabolomic tissue signature in human non-alcoholic fatty liver disease identifies protective candidate metabolites

Background Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries, yet its pathophysiology is incompletely understood. Small-molecule metabolite screens may offer new insights into disease mechanisms and reveal new treatment targets. Methods Discovery (N = 33) and replication (N = 66) of liver biopsies spanning the range from normal liver histology to non-alcoholic steatohepatitis (NASH) were ascertained ensuring rapid freezing under 30 s in patients. 252 metabolites were assessed using GC/MS. Replicated metabolites were evaluated in a murine high-fat diet model of NAFLD. Results In a two-stage metabolic screening, hydroquinone (HQ, pcombined = 3.0 × 10−4) and nicotinic acid (NA, pcombined = 3.9 × 10−9) were inversely correlated with histological NAFLD severity. A murine high-fat diet model of NAFLD demonstrated a protective effect of these two substances against NAFLD: Supplementation with 1% HQ reduced only liver steatosis, whereas 0.6% NA reduced both liver fat content and serum transaminase levels and induced a complex regulatory network of genes linked to NALFD pathogenesis in a global expression pathway analysis. Human nutritional intake of NA equivalent was also consistent with a protective effect of NA against NASH progression. Conclusion This first small-molecular screen of human liver tissue identified two replicated protective metabolites. Either the use of NA or targeting its regulatory pathways might be explored to treat or prevent human NAFLD.

Characterization and subchromosomal location of a novel tumor suppressor gene on human chromosome 7

Alterations in oncogenes and tumor suppressor genes (TSGs) are considered to be critical steps in oncogenesis. Consistent deletions and loss of heterozygosity (LOH) of polymorphic markers in a determinate chromosomal fragment are known to be indicative of a closely mapping TSG. Deletion of the long arm of chromosome 7 (hchr 7) is a frequent trait in many kinds of human primary tumors. LOH was studied with an extensive set of markers on chromosome 7q in several types of human neoplasias (primary breast, prostate, colon, ovarian and head and neck carcinomas) to determine the location of a putative TSG. The extent of LOH varied depending the type of tumor studied but all the LOH curves we obtained had a peak at (C-A)$\sb{\rm n}$ microsatellite repeat D7S522 at 7q31.1 and showed a Gaussian distribution. The high incidence of LOH in all tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on the 7q31.1. To investigate whether the putative TSG is conserved in the syntenic mouse locus, we studied LOH of 30 markers along mouse chromosome 6 (mchr 6) in chemically induced squamous cell carcinomas (SCCs). Tumors were obtained from SENCAR and C57BL/6 x DBA/2 F1 females by a two-stage carcinogenesis protocol. The high incidence of LOH in the tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on mchr 6 A1. Since this segment is syntenic with the hchr 7q31, these data indicate that the putative TSG is conserved in both species. Functional evidence for the existence of a TSG in hchr 7 was obtained by microcell fusion transfer of a single hchr 7 into a murine SCC-derived cell line. Five out of seven hybrids had two to three-fold longer latency periods for in vivo tumorigenicity assays than parental cells. One of the unrepressed hybrids had a deletion in the introduced chromosome 7 involving q31.1-q31.3, confirming the LOH data. ^

Home care service utilization in British Columbia: Substitute or complement to acute care?

The desire to promote efficient allocation of health resources and effective patient care has focused attention on home care as an alternative to acute hospital service. in particular, clinical home care is suggested as a substitute for the final days of hospital stay. This dissertation evaluates the relationship between hospital and home care services for residents of British Columbia, Canada beginning in 1993/94 using data from the British Columbia Linked Health database. ^ Lengths of stay for patients referred to home care following hospital discharge are compared to those for patients not referred to home care. Ordinary least squares regression analysis adjusts for age, gender, admission severity, comorbidity, complications, income, and other patient, physician, and hospital characteristics. Home care clients tend to have longer stays in hospital than patients not referred to home care (β = 2.54, p = 0.0001). Longer hospital stays are evident for all home care client groups as well as both older and younger patients. Sensitivity analysis for referral time to direct care and extreme lengths of stay are consistent with these findings. Two stage regression analysis indicates that selection bias is not significant.^ Patients referred to clinical home care also have different health service utilization following discharge compared to patients not referred to home care. Home care nursing clients use more medical services to complement home care. Rehabilitation clients initially substitute home care for physiotherapy services but later are more likely to be admitted to residential care. All home care clients are more likely to be readmitted to hospital during the one year follow-up period. There is also a strong complementary association between direct care referral and homemaker support. Rehabilitation clients have a greater risk of dying during the year following discharge. ^ These results suggest that home care is currently used as a complement rather than a substitute for some acute health services. Organizational and resource issues may contribute to the longer stays by home care clients. Program planning and policies are required if home care is to provide an effective substitute for acute hospital days. ^

Hormonal Contraception and the Risk of HIV Acquisition: An Individual Participant Data Meta-analysis.

BACKGROUND Observational studies of a putative association between hormonal contraception (HC) and HIV acquisition have produced conflicting results. We conducted an individual participant data (IPD) meta-analysis of studies from sub-Saharan Africa to compare the incidence of HIV infection in women using combined oral contraceptives (COCs) or the injectable progestins depot-medroxyprogesterone acetate (DMPA) or norethisterone enanthate (NET-EN) with women not using HC. METHODS AND FINDINGS Eligible studies measured HC exposure and incident HIV infection prospectively using standardized measures, enrolled women aged 15-49 y, recorded ≥15 incident HIV infections, and measured prespecified covariates. Our primary analysis estimated the adjusted hazard ratio (aHR) using two-stage random effects meta-analysis, controlling for region, marital status, age, number of sex partners, and condom use. We included 18 studies, including 37,124 women (43,613 woman-years) and 1,830 incident HIV infections. Relative to no HC use, the aHR for HIV acquisition was 1.50 (95% CI 1.24-1.83) for DMPA use, 1.24 (95% CI 0.84-1.82) for NET-EN use, and 1.03 (95% CI 0.88-1.20) for COC use. Between-study heterogeneity was mild (I2 < 50%). DMPA use was associated with increased HIV acquisition compared with COC use (aHR 1.43, 95% CI 1.23-1.67) and NET-EN use (aHR 1.32, 95% CI 1.08-1.61). Effect estimates were attenuated for studies at lower risk of methodological bias (compared with no HC use, aHR for DMPA use 1.22, 95% CI 0.99-1.50; for NET-EN use 0.67, 95% CI 0.47-0.96; and for COC use 0.91, 95% CI 0.73-1.41) compared to those at higher risk of bias (pinteraction = 0.003). Neither age nor herpes simplex virus type 2 infection status modified the HC-HIV relationship. CONCLUSIONS This IPD meta-analysis found no evidence that COC or NET-EN use increases women's risk of HIV but adds to the evidence that DMPA may increase HIV risk, underscoring the need for additional safe and effective contraceptive options for women at high HIV risk. A randomized controlled trial would provide more definitive evidence about the effects of hormonal contraception, particularly DMPA, on HIV risk.

Gender-Fair language and professional self-reference: The case of female psychologists in Polish

The struggle to achieve gender equality is accompanied by efforts to introduce gender-fair language. In languages with grammatical gender this implies the use of gender-appropriate forms (feminine for women and masculine for males). In the present research, results of a mixed method approach—a corpus analysis, a survey, and an experiment—provide consistent evidence that in Polish, feminine forms are still infrequent in women’s self-reference and that women psychologists continue to use masculine titles. Moreover, a qualitative inquiry examines the reasons why women prefer masculine over feminine job titles. Integrating findings from the two-stage design, we are able to identify the obstacles to promoting social change with the help of language and to understand the reasons behind them.

German Total Ankle Replacement Register of the German Foot and Ankle Society (D. A. F.) - presentation of design and reliability of the data as well as first results

INTRODUCTION Even though arthroplasty of the ankle joint is considered to be an established procedure, only about 1,300 endoprostheses are implanted in Germany annually. Arthrodeses of the ankle joint are performed almost three times more often. This may be due to the availability of the procedure - more than twice as many providers perform arthrodesis - as well as the postulated high frequency of revision procedures of arthroplasties in the literature. In those publications, however, there is often no clear differentiation between revision surgery with exchange of components, subsequent interventions due to complications and subsequent surgery not associated with complications. The German Orthopaedic Foot and Ankle Association's (D. A. F.) registry for total ankle replacement collects data pertaining to perioperative complications as well as cause, nature and extent of the subsequent interventions, and postoperative patient satisfaction. MATERIAL AND METHODS The D. A. F.'s total ankle replacement register is a nation-wide, voluntary registry. After giving written informed consent, the patients can be added to the database by participating providers. Data are collected during hospital stay for surgical treatment, during routine follow-up inspections and in the context of revision surgery. The information can be submitted in paper-based or online formats. The survey instruments are available as minimum data sets or scientific questionnaires which include patient-reported outcome measures (PROMs). The pseudonymous clinical data are collected and evaluated at the Institute for Evaluative Research in Medicine, University of Bern/Switzerland (IEFM). The patient-related data remain on the register's module server in North Rhine-Westphalia, Germany. The registry's methodology as well as the results of the revisions and patient satisfaction for 115 patients with a two year follow-up period are presented. Statistical analyses are performed with SAS™ (Version 9.4, SAS Institute, Inc., Cary, NC, USA). RESULTS About 2½ years after the register was launched there are 621 datasets on primary implantations, 1,427 on follow-ups and 121 records on re-operation available. 49 % of the patients received their implants due to post-traumatic osteoarthritis, 27 % because of a primary osteoarthritis and 15 % of patients suffered from a rheumatic disease. More than 90 % of the primary interventions proceeded without complications. Subsequent interventions were recorded for 84 patients, which corresponds to a rate of 13.5 % with respect to the primary implantations. It should be noted that these secondary procedures also include two-stage procedures not due to a complication. "True revisions" are interventions with exchange of components due to mechanical complications and/or infection and were present in 7.6 % of patients. 415 of the patients commented on their satisfaction with the operative result during the last follow-up: 89.9 % of patients evaluate their outcome as excellent or good, 9.4 % as moderate and only 0.7 % (3 patients) as poor. In these three cases a component loosening or symptomatic USG osteoarthritis was present. Two-year follow-up data using the American Orthopedic Foot and Ankle Society Ankle and Hindfoot Scale (AOFAS-AHS) are already available for 115 patients. The median AOFAS-AHS score increased from 33 points preoperatively to more than 80 points three to six months postoperatively. This increase remained nearly constant over the entire two-year follow-up period. CONCLUSION Covering less than 10 % of the approximately 240 providers in Germany and approximately 12 % of the annually implanted total ankle-replacements, the D. A. F.-register is still far from being seen as a national registry. Nevertheless, geographical coverage and inclusion of "high-" (more than 100 total ankle replacements a year) and "low-volume surgeons" (less than 5 total ankle replacements a year) make the register representative for Germany. The registry data show that the number of subsequent interventions and in particular the "true revision" procedures are markedly lower than the 20 % often postulated in the literature. In addition, a high level of patient satisfaction over the short and medium term is recorded. From the perspective of the authors, these results indicate that total ankle arthroplasty - given a correct indication and appropriate selection of patients - is not inferior to an ankle arthrodesis concerning patients' satisfaction and function. First valid survival rates can be expected about 10 years after the register's start.

Report of a European Society of Cardiology-European Association of Percutaneous Cardiovascular Interventions task force on the evaluation of coronary stents in Europe: executive summary.

The evaluation for European Union market approval of coronary stents falls under the Medical Device Directive that was adopted in 1993. Specific requirements for the assessment of coronary stents are laid out in supplementary advisory documents. In response to a call by the European Commission to make recommendations for a revision of the advisory document on the evaluation of coronary stents (Appendix 1 of MEDDEV 2.7.1), the European Society of Cardiology (ESC) and the European Association of Percutaneous Cardiovascular Interventions (EAPCI) established a Task Force to develop an expert advisory report. As basis for its report, the ESC-EAPCI Task Force reviewed existing processes, established a comprehensive list of all coronary drug-eluting stents that have received a CE mark to date, and undertook a systematic review of the literature of all published randomized clinical trials evaluating clinical and angiographic outcomes of coronary artery stents between 2002 and 2013. Based on these data, the TF provided recommendations to inform a new regulatory process for coronary stents. The main recommendations of the task force include implementation of a standardized non-clinical assessment of stents and a novel clinical evaluation pathway for market approval. The two-stage clinical evaluation plan includes recommendation for an initial pre-market trial with objective performance criteria (OPC) benchmarking using invasive imaging follow-up leading to conditional CE-mark approval and a subsequent mandatory, large-scale randomized trial with clinical endpoint evaluation leading to unconditional CE-mark. The data analysis from the systematic review of the Task Force may provide a basis for determination of OPC for use in future studies. This paper represents an executive summary of the Task Force's report.

A rapid and efficient ion-exchange chromatography for Lu–Hf, Sm–Nd, and Rb–Sr geochronology and the routine isotope analysis of sub-ng amounts of Hf by MC-ICP-MS

The development and improvement of MC-ICP-MS instruments have fueled the growth of Lu–Hf geochronology over the last two decades, but some limitations remain. Here, we present improvements in chemical separation and mass spectrometry that allow accurate and precise measurements of 176Hf/177Hf and 176Lu/177Hf in high-Lu/Hf samples (e.g., garnet and apatite), as well as for samples containing sub-nanogram quantities of Hf. When such samples are spiked, correcting for the isobaric interference of 176Lu on 176Hf is not always possible if the separation of Lu and Hf is insufficient. To improve the purification of Hf, the high field strength elements (HFSE, including Hf) are first separated from the rare earth elements (REE, including Lu) on a first-stage cation column modified after Patchett and Tatsumoto (Contrib. Mineral. Petrol., 1980, 75, 263–267). Hafnium is further purified on an Ln-Spec column adapted from the procedures of Münker et al. (Geochem., Geophys., Geosyst., 2001, DOI: 10.1029/2001gc000183) and Wimpenny et al. (Anal. Chem., 2013, 85, 11258–11264) typically resulting in Lu/Hf < 0.0001, Zr/Hf < 1, and Ti/Hf < 0.1. In addition, Sm–Nd and Rb–Sr separations can easily be added to the described two-stage ion-exchange procedure for Lu–Hf. The isotopic compositions are measured on a Thermo Scientific Neptune Plus MC-ICP-MS equipped with three 1012 Ω resistors. Multiple 176Hf/177Hf measurements of international reference rocks yield a precision of 5–20 ppm for solutions containing 40 ppb of Hf, and 50–180 ppm for 1 ppb solutions (=0.5 ng sample Hf 0.5 in ml). The routine analysis of sub-ng amounts of Hf will facilitate Lu–Hf dating of low-concentration samples.

The ROAM/EORTC-1308 trial: Radiation versus Observation following surgical resection of Atypical Meningioma: study protocol for a randomised controlled trial.

BACKGROUND Atypical meningiomas are an intermediate grade brain tumour with a recurrence rate of 39-58 %. It is not known whether early adjuvant radiotherapy reduces the risk of tumour recurrence and whether the potential side-effects are justified. An alternative management strategy is to perform active monitoring with magnetic resonance imaging (MRI) and to treat at recurrence. There are no randomised controlled trials comparing these two approaches. METHODS/DESIGN A total of 190 patients will be recruited from neurosurgical/neuro-oncology centres across the United Kingdom, Ireland and mainland Europe. Adult patients undergoing gross total resection of intracranial atypical meningioma are eligible. Patients with multiple meningioma, optic nerve sheath meningioma, previous intracranial tumour, previous cranial radiotherapy and neurofibromatosis will be excluded. Informed consent will be obtained from patients. This is a two-stage trial (both stages will run in parallel): Stage 1 (qualitative study) is designed to maximise patient and clinician acceptability, thereby optimising recruitment and retention. Patients wishing to continue will proceed to randomisation. Stage 2 (randomisation) patients will be randomised to receive either early adjuvant radiotherapy for 6 weeks (60 Gy in 30 fractions) or active monitoring. The primary outcome measure is time to MRI evidence of tumour recurrence (progression-free survival (PFS)). Secondary outcome measures include assessing the toxicity of the radiotherapy, the quality of life, neurocognitive function, time to second line treatment, time to death (overall survival (OS)) and incremental cost per quality-adjusted life year (QALY) gained. DISCUSSION ROAM/EORTC-1308 is the first multi-centre randomised controlled trial designed to determine whether early adjuvant radiotherapy reduces the risk of tumour recurrence following complete surgical resection of atypical meningioma. The results of this study will be used to inform current neurosurgery and neuro-oncology practice worldwide. TRIAL REGISTRATION ISRCTN71502099 on 19 May 2014.

Cultural Legacies and Political Preferences: The Failure of Separatism in the Swiss Jura

The study of secession generally stresses the causal influence of cultural identities, political preferences, or ecological factors. Whereas these different views are often considered to be mutually exclusive, this paper proposes a two-stage model in which they are complementary. We posit that cultural identities matter for explaining secessionism, but not because of primordial attachments. Rather, religious and linguistic groups matter because their members are imbued with cultural legacies that lead to distinct political preferences – in this case preferences over welfare statism. Further, ecological constraints such as geography and topography affect social interaction with like-minded individuals. On the basis of both these political preferences and ecological constraints, individuals then make rational choices about the desirability of secession. Instrumental considerations are therefore crucial in explaining the decision to secede, but not in a conventional pocketbook manner. To examine this theory, we analyze the 2013 referendum on the secession of the Jura Bernois region from the Canton of Berne in Switzerland, using municipal level census and referendum data. The results lend support to the theory and suggest one way in which the politics of identity, based on factors like language and religion, can be fused with the politics of interest (preferences for more or less state intervention into the polity and economy) to better understand group behavior.

Uric Acid and Cardiovascular Events: A Mendelian Randomization Study

Obesity and diets rich in uric acid-raising components appear to account for the increased prevalence of hyperuricemia in Westernized populations. Prevalence rates of hypertension, diabetes mellitus, CKD, and cardiovascular disease are also increasing. We used Mendelian randomization to examine whether uric acid is an independent and causal cardiovascular risk factor. Serum uric acid was measured in 3315 patients of the Ludwigshafen Risk and Cardiovascular Health Study. We calculated a weighted genetic risk score (GRS) for uric acid concentration based on eight uric acid-regulating single nucleotide polymorphisms. Causal odds ratios and causal hazard ratios (HRs) were calculated using a two-stage regression estimate with the GRS as the instrumental variable to examine associations with cardiometabolic phenotypes (cross-sectional) and mortality (prospectively) by logistic regression and Cox regression, respectively. Our GRS was not consistently associated with any biochemical marker except for uric acid, arguing against pleiotropy. Uric acid was associated with a range of prevalent diseases, including coronary artery disease. Uric acid and the GRS were both associated with cardiovascular death and sudden cardiac death. In a multivariate model adjusted for factors including medication, causal HRs corresponding to each 1-mg/dl increase in genetically predicted uric acid concentration were significant for cardiovascular death (HR, 1.77; 95% confidence interval, 1.12 to 2.81) and sudden cardiac death (HR, 2.41; 95% confidence interval, 1.16 to 5.00). These results suggest that high uric acid is causally related to adverse cardiovascular outcomes, especially sudden cardiac death.

Corruption and Growth Under Weak Identification

The goal of this paper is to revisit the influential work of Mauro [1995] focusing on the strength of his results under weak identification. He finds a negative impact of corruption on investment and economic growth that appears to be robust to endogeneity when using two-stage least squares (2SLS). Since the inception of Mauro [1995], much literature has focused on 2SLS methods revealing the dangers of estimation and thus inference under weak identification. We reproduce the original results of Mauro [1995] with a high level of confidence and show that the instrument used in the original work is in fact 'weak' as defined by Staiger and Stock [1997]. Thus we update the analysis using a test statistic robust to weak instruments. Our results suggest that under Mauro's original model there is a high probability that the parameters of interest are locally almost unidentified in multivariate specifications. To address this problem, we also investigate other instruments commonly used in the corruption literature and obtain similar results.

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^