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1.  TMEM14C is required for erythroid mitochondrial heme metabolism 
The Journal of Clinical Investigation  2014;124(10):4294-4304.
The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.
PMCID: PMC4191016  PMID: 25157825
2.  The Effect of Increasing Doses of Saw Palmetto Fruit Extract on Serum PSA Levels: Analysis of the CAMUS Randomized Trial 
The Journal of urology  2012;189(2):486-492.
Saw palmetto extracts are used for treating lower urinary tract symptoms in men despite level I evidence concluding that saw palmetto was ineffective in reducing lower urinary symptoms. We sought to determine whether higher doses of saw palmetto as studied in CAMUS affect serum PSA levels.
Materials and Methods
The CAMUS trial was a randomized, placebo-controlled double blind multi-centered North American trial conducted between June 5, 2008 and October 10, 2012 in which 369 men >45 years of age with AUA symptom score ≥ 8 and ≤ 24 were randomly assigned to placebo or dose escalation saw palmetto, which consisted of 320mg for first 24 weeks to 640mg for next 24 weeks to 960mg for last 24 weeks of this 72 week trial. Serum PSA levels (Beckman-Coulter) were obtained at baseline and at weeks 24, 48 and 72 and were compared between treatment groups using the pooled t and Fisher's exact tests.
Serum PSA levels were similar at baseline for the placebo (1.93 ± 1.59 ng/ml) and saw palmetto groups (2.20 ± 1.95, p = 0.16). Changes in PSA levels over the course of the study were similar: placebo group mean change 0.16 ± 1.08 ng/ml and saw palmetto group mean change 0.23 ± 0.83 ng/ml (p value 0.50). Additionally, no differential effect on serum PSA levels was observed between treatment arms when groups were stratified by baseline PSA values.
Saw palmetto extract does not affect serum PSA levels more than placebo even at relatively high doses.
PMCID: PMC3979451  PMID: 23253958
saw palmetto extract; Serenoa repens; prostate specific antigen; benign prostatic hyperplasia
3.  Magnetic Resonance Imaging as a Predictor of Pathologic Response in Patients Treated with Neoadjuvant Systemic Treatment for Operable Breast Cancer (TBCRC 017) 
Cancer  2013;119(10):1776-1783.
Increased pathologic complete response (pCR) rates observed with neoadjuvant chemotherapy (NCT) for some subsets of patients with invasive breast cancer has prompted interest in whether patients with pCR can be identified preoperatively and potentially spared the morbidity of surgery. This multicenter retrospective study was performed to estimate the accuracy of preoperative MRI in predicting pCR in the breast.
MRI at baseline and after completion of NCT plus data regarding pathologic response was collected retrospectively from 746 women treated at 8 institutions between 2002–2011. Tumors were characterized by immunohistochemical (IHC) phenotype into 4 categories based on receptor expression: hormone (estrogen & progesterone) receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2) negative (n=327), HR-positive, HER2-positive, (n=148), HR-negative, HER2-positive, (n=101), and triple-negative (HR-negative, HER2-negative, n=155). 194/249 (78%) patients with HER2-positive tumors received trastuzumab. Univariate and multivariate analyses of factors associated with radiographic complete response (rCR) and pCR were performed.
rCR and pCR for total group were 182/746 (24%) and 179/746 (24%), respectively, with the highest rate of pCR seen among triple-negative (57/155; 37%) and HER2 positive (38/101; 38%) subtypes. Overall accuracy of MRI for pCR prediction was 74%. Sensitivity, NPV, PPV, and accuracy differed significantly among tumor subtypes, with the greatest NPV in the TN (60%) and HER2 positive (62%) subtypes.
Overall accuracy of MRI for predicting pCR in invasive breast cancer patients receiving NCT was 74%. MR performance differed among subtypes possibly influenced by differences in pCR rates between groups. Future studies will determine whether MRI in combination with directed core biopsy improves predictive value for pathologic response.
PMCID: PMC3939707  PMID: 23436342
breast cancer; neoadjuvant chemotherapy; MRI; accuracy; pathologic complete response
4.  Relationships between Participants' International Prostate Symptom Score and BPH Impact Index Changes and Global Ratings of Change in a Trial of Phytotherapy for Men with Lower Urinary Tract Symptoms 
The Journal of urology  2012;189(3):987-992.
To relate changes in AUA Symptom Index (AUASI) scores with bother measures and global ratings of change among men with lower urinary tract symptoms enrolled in a trial of saw palmetto.
Materials and Methods
To be eligible, men were ≥45 years old, had ajpeak uroflow ≥4 ml/sec, and an AUASI score ≥ 8 and ≤ 24. Participants self-administered the AUASI, IPSS quality of life item (IPSS QoL), BPH Impact Index (BII) and two global change questions at baseline and 24, 48, and 72 weeks.
Among 357 participants, global ratings of “a little better” were associated with mean decreases in AUASI scores from 2.8 to 4.1 points, across three time points. The analogous range for mean decreases in BII scores was 1.0 to 1.7 points, and for the IPSS QoL item 0.5 to 0.8 points. At 72 weeks, for the first global change question, each change measure could discriminate between participants rating themselves at least a little better versus unchanged or worse 70-72% of the time. A multivariable model increased discrimination to 77%. For the second global change question, each change measure correctly discriminated ratings of at least a little better versus unchanged or worse 69-74% of the time, and a multivariable model increased discrimination to 79%.
Changes in AUASI scores could discriminate between participants rating themselves at least a little better versus unchanged or worse. Our findings support the practice of powering studies to detect group mean differences in AUASI scores of at least 3 points.
PMCID: PMC3932029  PMID: 23017510
lower urinary tract symptoms; health status index; psychometrics
5.  Harvest for Health Gardening Intervention Feasibility Study in Cancer Survivors 
Acta oncologica (Stockholm, Sweden)  2013;52(6):1110-1118.
Cancer survivors are at increased risk for second malignancies, cardiovascular disease, diabetes, and functional decline. Evidence suggests that a healthful diet and physical activity may reduce the risk of chronic disease and improve health in this population.
We conducted a feasibility study to evaluate a vegetable gardening intervention that paired 12 adult and child cancer survivors with Master Gardeners to explore effects on fruit and vegetable intake, physical activity, quality-of-life, and physical function. Throughout the year-long study period, the survivor-Master Gardener dyads worked together to plan/plant 3 gardens, harvest/rotate plantings, and troubleshoot/correct problems. Data on diet, physical activity, and quality-of-life were collected via surveys; anthropometrics and physical function were objectively measured. Acceptability of the intervention was assessed with a structured debriefing survey.
The gardening intervention was feasible (robust enrollment; minimal attrition) and well-received by cancer survivors and Master Gardeners. Improvement in 3 of 4 objective measures of strength, agility, and endurance was observed in 90% of survivors, with the following change scores (median [interquartile range]) noted between baseline and 1-year follow-up: hand grip test (+4.8 [3.0, 6.7] kg), 8 foot Get-Up-and-Go (−1.0 [−1.8, −0.2] seconds), 30-second chair stand (+3.0 [−1.0, 5.0] stands), and 6-minute walk (+38 [20, 160] feet). Increases of ≥1 fruit and vegetable serving/ day and ≥30 minutes/week of physical activity were observed in 40% and 60%, respectively.
These preliminary results support the feasibility and acceptability of a mentored gardening intervention and suggest that it may offer a novel and promising strategy to improve fruit and vegetable consumption, physical activity, and physical function in cancer survivors. A larger randomized controlled trial is needed to confirm our results.
PMCID: PMC3718632  PMID: 23438359
cancer survivors; gardening; intervention; health; diet
6.  Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation 
The Journal of Clinical Investigation  2013;123(8):3292-3304.
About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%–30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage–specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN–responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/β IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/β antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN–responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage–specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.
PMCID: PMC3726146  PMID: 23863621
7.  Prognostic Relevance of Cytochrome c Oxidase in Primary Glioblastoma Multiforme 
PLoS ONE  2013;8(4):e61035.
Patients with primary glioblastoma multiforme (GBM) have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO) promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%), and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.
PMCID: PMC3622606  PMID: 23593382
8.  Direct Recruitment of Polycomb Repressive Complex 1 (PRC1) to Chromatin by Core Binding Transcription Factors 
Molecular Cell  2012;45(3):330-343.
Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ, and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.
PMCID: PMC3278717  PMID: 22325351
9.  Preclinical Evaluation of Oncolytic Δγ134.5 Herpes Simplex Virus Expressing Interleukin-12 for Therapy of Breast Cancer Brain Metastases 
The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ134.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including “triple-negative” and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ134.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases.
PMCID: PMC3549352  PMID: 23346408
10.  Effect of Increasing Doses of Saw Palmetto on Lower Urinary Tract Symptoms: A Randomized Trial 
Jama  2011;306(12):1344-1351.
Saw palmetto fruit extracts are widely used for treating lower urinary tract symptoms attributed to benign prostatic hyperplasia. However, recent clinical trials have questioned their efficacy, at least at standard doses (320 mg daily).
To determine the effect of a saw palmetto extract at up to three times the standard dose on lower urinary tract symptoms attributed to benign prostatic hyperplasia.
Multicenter placebo-controlled randomized trial conducted from June, 2008 through October, 2010.
Eleven North American clinical sites.
Were men at least 45 years old, with a peak urinary flow rate ≥ 4 ml/sec, an AUA Symptom Index (AUASI) score ≥ 8 and ≤ 24, and no exclusions.
One, two, and then three 320 mg daily doses of saw palmetto extract or placebo, with dose increases at 24 and 48 weeks.
Main Outcome Measures
Primary outcome was the difference in AUASI score from baseline to 72 weeks. Secondary outcomes were measures of urinary bother; nocturia; uroflow; postvoid residual; prostate-specific antigen; participants’ global assessments; and indices of sexual function, continence, sleep quality, and prostatitis symptoms.
From baseline to 72 weeks, mean AUASI scores decreased from 14.4 to 12.2 points with saw palmetto and from 14.7 to 11.7 points with placebo. The group mean difference in AUASI score change from baseline to 72 weeks between the saw palmetto and placebo groups was 0.79 points favoring placebo (bound of the 95% confidence interval most favorable to saw palmetto was 1.77 points, one-sided P=0.91). Saw palmetto was no more effective than placebo for any secondary outcome. No attributable side effects were identified.
Increasing doses of a saw palmetto fruit extract did not reduce lower urinary tract symptoms more than placebo. (CAMUS study number NCT00603304
PMCID: PMC3326341  PMID: 21954478
11.  BRCA1 proteins regulate growth of ovarian cancer cells by tethering Ubc9 
Mutation in the BRCA1 gene is associated with increased risk for hereditary breast and ovarian cancers. In sporadic ovarian tumors, BRCA1 dysfunction is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein. Our group has previously reported that BRCA1 proteins, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme Ubc9. In this study, we examined the result of altered Ubc9 binding and knockdown on the sub-cellular localization and growth inhibitory function of BRCA1 proteins in ovarian cancer cells. Using live imaging of YFP, RFP-tagged BRCA1 and BRCA1a proteins, our results show enhanced cytoplasmic localization of K109R and C61G mutant BRCA1 proteins in ES-2, NIHOVCAR3 and UWB 1.289 ovarian cancer cells. Down-regulation of Ubc9 in ovarian cancer cells using Ubc9 siRNA resulted in cytoplasmic localization of BRCA1 and BRCA1a proteins. These mutant BRCA1a proteins were impaired in their capacity to inhibit growth of ES-2 ovarian cancer cells. Several ovarian cancer cells, including a BRCA1-null ovarian cancer cell line, showed higher levels of expression of Ubc9. This is the first study demonstrating the physiological link between loss of Ubc9 binding and loss of growth suppression of disease-associated mutant BRCA1a proteins in ovarian cancer cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of ovarian cancers.
PMCID: PMC3433105  PMID: 22957306
BRCA1; BRCA1a; Ubc9; Ovarian cancer; RING domain mutants; nuclear import; Growth suppression
12.  Improved survival in BRCA2 carriers with ovarian cancer 
Familial Cancer  2007;6(1):113-119.
The objective of this study was to investigate survival of ovarian cancer patients with BRCA1 and BRCA2 mutations compared to those without mutations in a population-based sample of incident epithelial ovarian cancer cases.
Follow-up for vital status was performed on a population-based sample of 232 women with incident epithelial ovarian cancer recruited between December 13, 2000 and September 30, 2003 in the Tampa Bay area. Survival analysis using Cox regression was performed on (1) all 232 cases and (2) the 209 invasive epithelial ovarian cancer cases. Results of the two analyses were similar, thus data involving the 209 invasive epithelial cancer cases are presented, as this was judged to be more clinically relevant.
In the multivariate analysis, BRCA status and stage were statistically significant, and were adjusted for in the survival analysis model. The Kaplan–Meier method estimated expected survival at 4 years of 83% of BRCA2 carriers compared to 37% of BRCA1 carriers and 12% of non-carriers. There was a statistically significant difference between BRCA2 carriers and non-carriers (p = 0.013). No statistically significant survival differences were seen for BRCA1 carriers when compared with either BRCA2 carriers or non-carriers.
These data suggest that BRCA2 mutation carriers with ovarian cancer may have better survival than BRCA1 carriers and non-carriers. The etiology of this possible survival advantage is currently unknown. Larger studies are needed to confirm these results and to clarify their etiology and clinical significance.
PMCID: PMC3303221  PMID: 17160431
hereditary ovarian cancer carcinoma; BRCA1; BRCA2; Survival
13.  Identification of Distal cis-Regulatory Elements at Mouse Mitoferrin Loci Using Zebrafish Transgenesis▿ 
Molecular and Cellular Biology  2011;31(7):1344-1356.
Mitoferrin 1 (Mfrn1; Slc25a37) and mitoferrin 2 (Mfrn2; Slc25a28) function as essential mitochondrial iron importers for heme and Fe/S cluster biogenesis. A genetic deficiency of Mfrn1 results in a profound hypochromic anemia in vertebrate species. To map the cis-regulatory modules (CRMs) that control expression of the Mfrn genes, we utilized genome-wide chromatin immunoprecipitation (ChIP) datasets for the major erythroid transcription factor GATA-1. We identified the CRMs that faithfully drive the expression of Mfrn1 during blood and heart development and Mfrn2 ubiquitously. Through in vivo analyses of the Mfrn-CRMs in zebrafish and mouse, we demonstrate their functional and evolutionary conservation. Using knockdowns with morpholinos and cell sorting analysis in transgenic zebrafish embryos, we show that GATA-1 directly regulates the expression of Mfrn1. Mutagenesis of individual GATA-1 binding cis elements (GBE) demonstrated that at least two of the three GBE within this CRM are functionally required for GATA-mediated transcription of Mfrn1. Furthermore, ChIP assays demonstrate switching from GATA-2 to GATA-1 at these elements during erythroid maturation. Our results provide new insights into the genetic regulation of mitochondrial function and iron homeostasis and, more generally, illustrate the utility of genome-wide ChIP analysis combined with zebrafish transgenesis for identifying long-range transcriptional enhancers that regulate tissue development.
PMCID: PMC3135305  PMID: 21248200
14.  Rationally designed pharmacogenomic treatment using concurrent capecitabine and radiotherapy for glioblastoma; gene expression profiles associated with outcome 
Previous preclinical studies suggested that concurrent capecitabine and radiation could be an effective new treatment modality for glioblastoma (GBM). In the current study we investigate toxicity and response to this regimen and explore associations between gene expression and patient outcome.
Experimental Design
Eighteen newly diagnosed GBM patients received concurrent capecitabine at 625 mg/m2 BID (25% escalation) and irradiation (60 Gy total) for 6 weeks followed by 4 weeks of capecitabine only. Maintenance capecitabine was administered for 14 days every 3 weeks until progression or unacceptable toxicity. Expression analysis of 94 genes involved in capecitabine metabolism and radiation response was performed on tissues obtained prior to therapy. The relationship between gene expression with time-to-progression (TTP) and overall survival (OS) was investigated using univariate Cox proportional hazards regression, semi-supervised principle component analysis (SSPCA), and class prediction modeling.
The maximum tolerated dose of capecitabine was 625 mg/m2 BID. Median patient TTP and OS were 247 and 367 days respectively. Cox regression identified 24 genes significantly (p<0.025) associated with patient outcome. SSPCA analysis identified two patient populations significantly different in both TTP (p=0.005) and OS (p=0.015). Class prediction modeling determined that 8 genes (RAD54B, MTOR, DCTD, APEX2, TK1, RRM2, SLC29A1, ERCC6) could collectively classify patients into outcome subgroups with 100% accuracy and precision.
Capecitabine and concurrent radiation for newly diagnosed GBM appears well tolerated and comparable to temozolomide and radiation. A gene expression profile predictive of patient outcome that may be useful in patient stratification for therapy was also elucidated.
PMCID: PMC2871063  PMID: 20460474
15.  A Myc rather than core pluripotency module accounts for the shared signatures of embryonic stem and cancer cells 
Cell  2010;143(2):313-324.
c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies, and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc), and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is comprised of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects in large part the pervasive nature of Myc regulatory network.
PMCID: PMC3018841  PMID: 20946988
16.  TIF1γ controls erythroid cell fate by regulating transcription elongation 
Cell  2010;142(1):133-143.
Recent genome-wide studies have demonstrated pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1γ mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1γ-deficient animals. Biochemical analysis established physical interactions among TIF1γ, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. ChIP assays in human CD34+ cells supported a TIF1γ-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.
PMCID: PMC3072682  PMID: 20603019
17.  Common Features of Megakaryocytes and Hematopoietic Stem Cells: What's the Connection? 
Journal of cellular biochemistry  2009;107(5):857-864.
Megakaryocytes are rare polyploid bone marrow cells whose function is to produce blood platelets. Since the purification and cloning of the major megakaryocyte cytokine, thrombopoietin, in 1994, considerable progress has been made in understanding the biology of megakaryocyte development. Remarkably, these advances have revealed a number of key features of megakaryocytes that are shared with hematopoietic stem cells (HSCs), such as common surface receptors, lineage-specific transcription factors, and specialized signaling pathways. Why there should be such a close connection between these two cell types remains unclear. In this Prospects article, we summarize the data supporting these shared features and speculate on possible teleological bases. In particular, we focus on common links involving developmental hierarchy, endothelial cells, and bone marrow niche interactions. This discussion highlights new data showing close ontologic relationship between HSCs and specialized “hemogenic” endothelial cells during development, and functional overlap between megakaryocytes/platelets and endothelial cells. Overall, these findings may be of relevance in the development of techniques for HSC ex vivo culture and/or possible generation of HSCs via somatic cell reprogramming.
PMCID: PMC2741141  PMID: 19492306
18.  Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis 
Molecular cell  2009;36(4):682-695.
The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo.
PMCID: PMC2800995  PMID: 19941827
GATA-1; Polycomb; Zbtb7a; erythroid; ChIP-seq
19.  Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation 
Current protocols in stem cell biology  2009;CHAPTER:Unit1B.5.
In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification.
PMCID: PMC2782545  PMID: 19306258
tandem affinity purification; in vivo biotinylation; protein-protein interaction; embryonic stem cells
20.  Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development▿ §  
Molecular and Cellular Biology  2009;29(15):4103-4115.
The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.
PMCID: PMC2715817  PMID: 19470763
21.  TLX1 (HOX11) Immortalization of Embryonic Stem Cell-Derived and Primary Murine Hematopoietic Progenitors 
Current protocols in stem cell biology  2008;CHAPTER:Unit-1F.7.
The ability to generate genetically engineered cell lines is of great experimental value. They provide a renewable source of material that may be suitable for biochemical analyses, chromatin immunoprecipitation assays, structure-function studies, gene function assignment, and transcription factor target gene identification. This unit describes protocols for TLX1 (HOX11)-mediated immortalization of murine hematopoietic progenitors derived from in vitro differentiated murine embryonic stem cells, or from primary mouse fetal liver or bone marrow. A wide variety of hematopoietic cell types have been immortalized using these procedures including erythroid, megakaryocytic, monocytic, myelocytic, and multipotential cell types. These lines are typically cytokine dependent for their survival and growth.
PMCID: PMC2633409  PMID: 19085976
hematopoietic cell immortalization; TLX1; HOX11; hematopoietic in vitro ES cell differentiation; hematopoietic progenitors
22.  Clinical Efficacy and Predictive Molecular Markers of Neoadjuvant Gemcitabine and Pemetrexed in Resectable Non-Small-Cell Lung Cancer 
A trial of neoadjuvant gemcitabine and pemetrexed (GP) chemotherapy in patients with resectable non-small cell lung cancer was conducted. The goal was to achieve a disease response rate of 50% and to determine if the expression levels of genes associated with gemcitabine and pemetrexed metabolism are predictive of response.
Patients had staging with a computed tomography (CT) scan, whole body F-18 fluorodeoxyglucose positron emission tomography (PET), and mediastinoscopy. Four biweekly cycles of GP were given. Patients were restaged, and those with resectable stage IB-III disease had thoracotomy. Fresh frozen tumor specimens were collected before and after chemotherapy and the mRNA levels of 14 target genes determined by real-time reverse transcriptase polymerase chain reaction (RTPCR).
Fifty-two patients started therapy. The radiographic disease response rate was 35% (95% confidence interval [CI] 21.7 to 49.6%), and the progression rate was 6%. Forty-six patients had a thoracotomy. The complete tumor resection rate was 77% (40/52). There were no perioperative deaths or deaths related to chemotherapy. Tumor response to chemotherapy was inversely correlated with the level of expression of RRM1 (p ≤ 0.001; regulatory subunit of ribonucleotide reductase) and TS (p = 0.006; thymidylate synthase); i.e., the reduction in tumor size was greater in those with low levels of expression.
Neoadjuvant GP is well tolerated and produces an objective response rate of 35%. Tumoral RRM1 and TS mRNA levels are predictive of disease response and should be considered as parameters for treatment selection in future trials with this regimen.
PMCID: PMC2639211  PMID: 18827606
Gemcitabine; Pemetrexed; Ribonucleotide Reductase; Thymidylate Synthase; Non-Small Cell Lung Cancer
23.  Antagonism of FOG-1 and GATA factors in fate choice for the mast cell lineage 
The zinc finger transcription factor GATA-1 requires direct physical interaction with the cofactor friend of GATA-1 (FOG-1) for its essential role in erythroid and megakaryocytic development. We show that in the mast cell lineage, GATA-1 functions completely independent of FOG proteins. Moreover, we demonstrate that FOG-1 antagonizes the fate choice of multipotential progenitor cells for the mast cell lineage, and that its down-regulation is a prerequisite for mast cell development. Remarkably, ectopic expression of FOG-1 in committed mast cell progenitors redirects them into the erythroid, megakaryocytic, and granulocytic lineages. These lineage switches correlate with transcriptional down-regulation of GATA-2, an essential mast cell GATA factor, via switching of GATA-1 for GATA-2 at a key enhancer element upstream of the GATA-2 gene. These findings illustrate combinatorial control of cell fate identity by a transcription factor and its cofactor, and highlight the role of transcriptional networks in lineage determination. They also provide evidence for lineage instability during early stages of hematopoietic lineage commitment.
PMCID: PMC2275384  PMID: 18299398
24.  Identification of ZBP-89 as a Novel GATA-1-Associated Transcription Factor Involved in Megakaryocytic and Erythroid Development▿ †  
Molecular and Cellular Biology  2008;28(8):2675-2689.
A complete understanding of the transcriptional regulation of developmental lineages requires that all relevant factors be identified. Here, we have taken a proteomic approach to identify novel proteins associated with GATA-1, a lineage-restricted zinc finger transcription factor required for terminal erythroid and megakaryocytic maturation. We identify the Krüppel-type zinc finger transcription factor ZBP-89 as being a component of multiprotein complexes involving GATA-1 and its essential cofactor Friend of GATA-1 (FOG-1). Using chromatin immunoprecipitation assays, we show that GATA-1 and ZBP-89 cooccupy cis-regulatory elements of certain erythroid and megakaryocyte-specific genes, including an enhancer of the GATA-1 gene itself. Loss-of-function studies in zebrafish and mice demonstrate an in vivo requirement for ZBP-89 in megakaryopoiesis and definitive erythropoiesis but not primitive erythropoiesis, phenocopying aspects of FOG-1- and GATA-1-deficient animals. These findings identify ZBP-89 as being a novel transcription factor involved in erythroid and megakaryocytic development and suggest that it serves a cooperative function with GATA-1 and/or FOG-1 in a developmental stage-specific manner.
PMCID: PMC2293107  PMID: 18250154
25.  Loss of Nuclear p21Cip1/WAF1 During Neoplastic Progression to Metastasis in γ-Irradiated p21 Hemizygous Mice 
p21Cip1/WAF1 localizes to the nucleus in response to γ-irradiation induced DNA damage and mediates a G1 checkpoint arrest. Although γ-irradiated p21+/- mice develop a broad spectrum of tumors, γ-irradiated p21-/- mice develop significantly more metastatic cancers. To evaluate the expression of p21 in tissues prone or resistant to tumorigenesis as a function of γ-irradiation, and to determine whether phenotypic loss of p21 heterozygosity occurs during tumor progression in p21+/- mice, tissues and tumors from γ-irradiated mice were evaluated immunohistochemically. The percentage of tumors in p21+/- mice that were nuclear p21-positive declined with progression to metastasis (p<0.0001). Benign tumors were more often p21-positive and comprised of larger subsets of nuclear p21-positive cells than were malignant tumors of the same histopathological type, while metastatic cancers were nuclear p21-negative (p=0.0003). Even when a primary cancer was comprised of a subset of nuclear p21-positive cells, the metastatic foci of that same cancer were nuclear p21-negative. Mesenchymal tumors, though rare, were more likely metastatic than were epithelial tumors (p=0.0004), and these were invariably nuclear p21-negative. Prepubescent epithelial tissues from which most tumors later originated in mice with reduced p21 gene dosage, (i.e., harderian gland, ovary, small intestine, and lung) were p21 expressive within 4 hours of γ-irradiation (p=0.0625), so that p21/Ki67 ratios increased post-γ-irradiation (p=0.03). In contrast, p21 did not localize to nuclei of cortical thymocytes, a tissue where tumorigenesis was not augmented by reduced p21 gene dosage. Cellular subclones of malignant tumors, especially those of mesenchymal cell origin, which lack nuclear p21 may more readily acquire the genetic alterations of the metastatic phenotype.
PMCID: PMC2039892  PMID: 17207793
p21Cip1/WAF1; p21 knockout mice; metastasis; immunohistochemistry; tumorigenesis

Results 1-25 (31)