Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins.

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37 degrees C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1Delta temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1Delta cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

Hormone therapy and physical activity as predictors of lung cancer risk and survival: The California Teachers Study

Cigarette smoking is responsible for the majority of lung cancer cases worldwide; however, a proportion of never smokers still develop lung cancer over their lifetime, prompting investigation into additional factors that may modify lung cancer incidence, as well as mortality. Although hormone therapy (HT), physical activity (PA), and lung cancer have been previously examined, the associations remain unclear. This study investigated exposure to HT and PA that may modulate underlying mechanisms of lung cancer etiology and progression among women by using existing, de-identified data from the California Teachers Study (CTS).^ The CTS cohort, established in 1995–1996, has 133,479 active and retired female teachers and administrators, recruited through the California State Teachers Retirement System, and followed annually for cancer diagnosis, death, and change of address. Each woman enrolled in the CTS returned a questionnaire covering a wide variety of issues related to cancer risk and women's health, including recent and past HT use and physical activity, as well as active and environmental cigarette smoke exposure. Complete data to assess the associations between HT and lung cancer risk and survival were available for 60,592 postmenopausal women. Between 1995 and 2007, 727 of these women were diagnosed with invasive lung cancer; 441 of these died. Complete data to assess the associations between PA and lung cancer risk and survival were available for 118,513 women. Between 1995 and 2007, 853 of these women were diagnosed with invasive lung cancer; 516 of these died.^ After careful adjustment for smoking habits and other potential confounders, no measure of HT use was associated with lung cancer risk; however, any HT use (vs. no use) was associated with a decrease in lung-cancer-specific mortality. Specifically, among women who only used estrogen (E-only), decreases in lung cancer mortality were seen for recent use, but not for former use; no association was observed for estrogen plus progestin (E+P). Furthermore, among former users of HT, a statistically significant decrease in lung cancer mortality was observed for E-only use within 5 years prior to baseline, but not for E-only use >5 years prior to baseline. Neither long-term recreational PA nor recent recreational PA alone were associated with lung cancer risk; however, among women with a BMI<25 and ever smokers, high long-term moderate+strenuous PA was associated with a decrease in lung cancer risk. Women with non-local disease showed a decrease in lung cancer mortality associated with increasing duration of strenuous long-term activity, and 1.50-3.00 h/wk/y of recent moderate or recent strenuous PA. Long-term moderate PA was associated with decreased lung cancer mortality in never smokers, whereas recent moderate PA was associated with increased lung cancer mortality in current smokers. ^ Placing our findings in the context of the current literature, HT does not appear to be associated with lung cancer risk and previous studies reporting a protective effect of HT use on lung cancer risk may be subject to residual confounding by smoking. Looking at our findings regarding PA overall, the evidence still remains inconclusive regarding whether or not physical activity influence lung cancer risk or mortality. Our results suggest that recreational PA may associated with decreased lung cancer risk among women with BMI<25 and ever smoking-women; however, residual confounding by smoking should be strongly considered. To our knowledge, this is the first study to investigate lifetime recreational PA and lung cancer mortality among women. Our results contribute to the growing body of knowledge regarding non-smoking-related risk factors for lung cancer incidence and mortality among women. Given the potential clinical and interventional significance, further study and validation of these findings is warranted.^

The Effect of Proximity, Explicitness, and Representation of Basic Science Information on Student Clinical Problem-Solving

Problem: Medical and veterinary students memorize facts but then have difficulty applying those facts in clinical problem solving. Cognitive engineering research suggests that the inability of medical and veterinary students to infer concepts from facts may be due in part to specific features of how information is represented and organized in educational materials. First, physical separation of pieces of information may increase the cognitive load on the student. Second, information that is necessary but not explicitly stated may also contribute to the student’s cognitive load. Finally, the types of representations – textual or graphical – may also support or hinder the student’s learning process. This may explain why students have difficulty applying biomedical facts in clinical problem solving. Purpose: To test the hypothesis that three specific aspects of expository text – the patial distance between the facts needed to infer a rule, the explicitness of information, and the format of representation – affected the ability of students to solve clinical problems. Setting: The study was conducted in the parasitology laboratory of a college of veterinary medicine in Texas. Sample: The study subjects were a convenience sample consisting of 132 second-year veterinary students who matriculated in 2007. The age of this class upon admission ranged from 20-52, and the gender makeup of this class consisted of approximately 75% females and 25% males. Results: No statistically significant difference in student ability to solve clinical problems was found when relevant facts were placed in proximity, nor when an explicit rule was stated. Further, no statistically significant difference in student ability to solve clinical problems was found when students were given different representations of material, including tables and concept maps. Findings: The findings from this study indicate that the three properties investigated – proximity, explicitness, and representation – had no statistically significant effect on student learning as it relates to clinical problem-solving ability. However, ad hoc observations as well as findings from other researchers suggest that the subjects were probably using rote learning techniques such as memorization, and therefore were not attempting to infer relationships from the factual material in the interventions, unless they were specifically prompted to look for patterns. A serendipitous finding unrelated to the study hypothesis was that those subjects who correctly answered questions regarding functional (non-morphologic) properties, such as mode of transmission and intermediate host, at the family taxonomic level were significantly more likely to correctly answer clinical case scenarios than were subjects who did not correctly answer questions regarding functional properties. These findings suggest a strong relationship (p < .001) between well-organized knowledge of taxonomic functional properties and clinical problem solving ability. Recommendations: Further study should be undertaken investigating the relationship between knowledge of functional taxonomic properties and clinical problem solving ability. In addition, the effect of prompting students to look for patterns in instructional material, followed by the effect of factors that affect cognitive load such as proximity, explicitness, and representation, should be explored.