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

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

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

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant

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 retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1δG) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1δG/δG mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1δG/δG MEFs display a magnitude of E2F target gene derepression similar to that of Rb1-/- cells, even though Rb1δG/δG cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1δG/δG MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1δG/δG mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non- E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified. © 2014, American Society for Microbiology

Toward Return to Duty Decision-Making After Military Mild Traumatic Brain Injury: Preliminary Validation of the Charge of Quarters Duty Test

Determining duty-readiness after mild traumatic brain injury (mTBI) remains a priority of the United States Department of Defense as warfighters in both deployed and non-deployed settings continue to sustain these injuries in relatively large numbers. Warfighters with mTBI may experience unresolved sensorimotor, emotional, cognitive sequelae including problems with executive functions, a category of higher order cognitive processes that enable people to regulate goal-directed behavior. Persistent mTBI sequelae interfere with warfighters’ proficiency in performing military duties and signal the need for graded return to activity and possibly rehabilitative services. Although significant strides have been carried out in recent years to enhance the identification and management of mTBI in garrison (EXORD 165–13) and deployed settings (EXORD 242–11; DoDI 6,490.11), the Department of Defense still lacks reliable, valid, and clinically feasible functional assessments to help inform duty-readiness decisions. Traditional functional assessments lack face validity for warfighters and may have ceiling effects, especially as related to executive functions. Performance-based multitasking assessments have been shown to be sensitive to executive dysfunction after acquired brain injury but no multitasking assessments have been validated in adults with mTBI. Existing multitasking assessments are not ecologically valid relative to military contexts. A multidisciplinary military–civilian team of researchers developed and evaluated a performance-based assessment called the Assessment of Military Multitasking Performance. One of the Assessment of Military Multitasking Performance multitasks, the Charge of Quarters Duty Test (CQDT), was designed to challenge the divided attention, foresight, and planning dimensions of executive functions. Here, we report on the preliminary validation results of the CQDT