Dissecting the molecular role of distinct binding interfaces on the retinoblastoma tumor suppressor in growth control and tumorigenesis.

The retinoblastoma tumor suppressor protein (pRB) functions to maintain proliferative control and act as a barrier to tumorigenesis. pRB is capable of regulating E2F transcription factors to mediate control of proliferation through transcriptional regulation of S-phase target gene expression. In addition, pRB can stabilize the CDK inhibitor p27 through an interaction with two ubiquitin ligase complexes. Further, pRB is capable of forming a unique interaction with E2F1 termed the ‘specific’ interaction that is capable of blocking E2F1 induced apoptosis. These functions of pRB are mediated by distinct binding interfaces and their contributions to the overall functionality of pRB are not well defined. In this thesis multiple experimental approaches are employed to study the function of the distinct binding sites in isolation to better define their functional roles. As described in chapter 2 the E2F1 ‘specific site’ is capable of maintaining and interaction with hyperphosphorylated pRB while the E2F ‘general site’ is disrupted by phosphorylation. This suggests that pRB can function beyond the G1 phase of the cell cycle to regulate E2F1 through the ‘specific site’. Using a series of novel synthetic mutations of pRB we found that multiple binding sites contribute in a redundant manner to the overall cell cycle arrest ability of pRB. While, the ‘general site’ appears to play a critical role in the regulation of cell cycle arrest through the regulation of E2F transcription factors, the LXCXE binding cleft and the ‘specific site’ can function redundantly to control proliferation. A gene-targeted mouse model was developed that disrupted the ‘general site’ while leaving other binding sites on pRB intact. Strikingly, these mice are unable to regulate E2F target gene expression yet they maintain appropriate proliferative control in multiple cellular contexts. The maintained proliferative control by pRB appears to be largely due to the activity of p27 as disruption of E2F regulation and p27 deficiency results in loss of proliferative control and subsequent tumorigenesis. Taken together, this work defines the contribution of the distinct binding sites to the overall functionality of pRB and provides insight into the disruption of pRB in human cancer

Analysis of cell cycle position in mammalian cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments. DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases. In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control. © 2012 Creative Commons Attribution License

Context dependent roles for RB-E2F transcriptional regulation in tumor suppression

RB-E2F transcriptional control plays a key role in regulating the timing of cell cycle progression from G1 to S-phase in response to growth factor stimulation. Despite this role, it is genetically dispensable for cell cycle exit in primary fibroblasts in response to growth arrest signals. Mice engineered to be defective for RB-E2F transcriptional control at cell cycle genes were also found to live a full lifespan with no susceptibility to cancer. Based on this background we sought to probe the vulnerabilities of RB-E2F transcriptional control defects found in Rb1 R461E,K542E mutant mice (Rb1 G ) through genetic crosses with other mouse strains. We generated Rb1 G/G mice in combination with Trp53 and Cdkn1a deficiencies, as well as in combination with Kras G12D . The Rb1 G mutation enhanced Trp53 cancer susceptibility, but had no effect in combination with Cdkn1a deficiency or Kras G12D . Collectively, this study indicates that compromised RB-E2F transcriptional control is not uniformly cancer enabling, but rather has potent oncogenic effects when combined with specific vulnerabilities

Interchangeable roles for E2F transcriptional repression by the retinoblastoma protein and p27\u3csup\u3eKIP1\u3c/sup\u3e-cyclindependent kinase regulation in cell cycle control and tumor suppression

The mammalian G1-S phase transition is controlled by the opposing forces of cyclin-dependent kinases (CDK) and the retinoblastoma protein (pRB). Here, we present evidence for systems-level control of cell cycle arrest by pRB-E2F and p27-CDK regulation. By introducing a point mutant allele of pRB that is defective for E2F repression (Rb1G) into a p27KIP1 null background (Cdkn1b-/-), both E2F transcriptional repression and CDK regulation are compromised. These double-mutant Rb1G/G; Cdkn1b-/- mice are viable and phenocopy Rb1+/- mice in developing pituitary adenocarcinomas, even though neither single mutant strain is cancer prone. Combined loss of pRB-E2F transcriptional regulation and p27KIP1 leads to defective proliferative control in response to various types of DNA damage. In addition, Rb1G/G; Cdkn1b-/- fibroblasts immortalize faster in culture and more frequently than either single mutant genotype. Importantly, the synthetic DNA damage arrest defect caused by Rb1G/G; Cdkn1b-/- mutations is evident in the developing intermediate pituitary lobe where tumors ultimately arise. Our work identifies a unique relationship between pRB-E2F and p27-CDK control and offers in vivo evidence that pRB is capable of cell cycle control through E2F-independent effects

Adenovirus E1A directly targets the E2F/DP-1 complex

Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection. © 2011, American Society for Microbiology

Multiple molecular interactions redundantly contribute to RB-mediated cell cycle control

Background: The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle. Results: Here we use a structure-function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB-E2F interactions are competitive with pRB-CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB\u27s ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver. Conclusions: Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network

Clinical Utility and Analysis of the Run-Roll-Aim Task: Informing Return-to-Duty Readiness Decisions in Active-Duty Service Members

Introduction The Assessment of Military Multitasking Performance (AMMP1) consists of six dual-task and multitask military-relevant performance-based assessments which were developed to provide assistance in making return-to-duty decisions after concussion or mild traumatic brain injury (mTBI.) The Run-Roll-Aim (RRA) task, one component of the AMMP, was developed to target vulnerabilities following mTBI including attention, visual function, dynamic stability, rapid transition, and vestibular function. One aim of this study was to assess the known-group and construct validity of the RRA, and additionally to further explore reliability limitations reported previously. Materials and Methods A cross-sectional study consisting of 84 Active Duty service members in two groups (healthy control – HC and individuals experiencing persistent mTBI symptoms) completed neurocognitive tests and the RRA. The RRA task requires a high level of mobility and resembles military training activities in a maneuver that includes combat rolls, fast transitions, obstacle avoidance, and visual search. Observational and inertial sensor data were compared between groups and performance across four trial times was compared within groups. Correlations between RRA results and neurocognitive test scores were analyzed. Results Simple observational measures (time, errors) did not differ between groups. Spectral power analysis of the inertial sensor data showed significant differences in motor performance between groups. Within group one-way ANOVAs showed that in HC trial 1, time was significantly different than trials 2,3 and 4 (F(3,47) = 4.60, p < 0.01, Tukey HSD p < 0.05) while the mTBI group showed no significant difference in time between trials. During testing individuals with mTBI were less likely to complete the multiple test trials or required additional rest between trials than HCs (χ2 = 10.78, p < 0.01). Small but significant correlations were seen with two neurocognitive tests of attention and RRA performance time. Conclusion While observational scores were not sensitive to group differences, inertial sensor data showed motor performance on the forward run, combat roll, and backward run differed significantly between groups. The RRA task appeared challenging and provoked symptoms in the mTBI group, causing 8 of 33 mTBI participants to stop the task or require additional rest between trials while none of the HC participants had to stop. Individuals with mTBI demonstrated slower learning of the complex motor sequence compared to HCs who had significant improvement after one trial of RRA. Complex novel training maneuvers like RRA may aid clinicians in informing return to duty decisions

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues

Measuring Soldier Performance During the Patrol-Exertion Multitask: Preliminary Validation of a Postconcussive Functional Return-to-Duty Metric

Objective: To assess the discriminant validity of the Patrol-Exertion Multitask (PEMT), a novel, multidomain, functional return-to-duty clinical assessment for active duty military personnel