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1.  Targeted Inhibition of ATR or CHEK1 Reverses Radioresistance in Oral Squamous Cell Carcinoma Cells with Distal Chromosome Arm 11q Loss 
Genes, chromosomes & cancer  2013;53(2):129-143.
Oral squamous cell carcinoma (OSCC), a subset of head and neck squamous cell carcinoma (HNSCC), is the eighth most common cancer in the U.S.. Amplification of chromosomal band 11q13 and its association with poor prognosis has been well established in OSCC. The first step in the breakage-fusion-bridge (BFB) cycle leading to 11q13 amplification involves breakage and loss of distal 11q. Distal 11q loss marked by copy number loss of the ATM gene is observed in 25% of all Cancer Genome Atlas (TCGA) tumors, including 48% of HNSCC. We showed previously that copy number loss of distal 11q is associated with decreased sensitivity (increased resistance) to ionizing radiation (IR) in OSCC cell lines. We hypothesized that this radioresistance phenotype associated with ATM copy number loss results from upregulation of the compensatory ATR-CHEK1 pathway, and that knocking down the ATR-CHEK1 pathway increases the sensitivity to IR of OSCC cells with distal 11q loss. Clonogenic survival assays confirmed the association between reduced sensitivity to IR in OSCC cell lines and distal 11q loss. Gene and protein expression studies revealed upregulation of the ATR-CHEK1 pathway and flow cytometry showed G2-M checkpoint arrest after IR treatment of cell lines with distal 11q loss. Targeted knockdown of the ATR-CHEK1 pathway using CHEK1 or ATR siRNA or a CHEK1 small molecule inhibitor (SMI, PF-00477736) resulted in increased sensitivity of the tumor cells to IR. Our results suggest that distal 11q loss is a useful biomarker in OSCC for radioresistance that can be reversed by ATR-CHEK1 pathway inhibition.
PMCID: PMC4216593  PMID: 24327542
2.  Kinesin molecular motor Eg5 functions during polypeptide synthesis 
Molecular Biology of the Cell  2011;22(18):3420-3430.
The microtubule motor Eg5 is well known for its functions during mitosis. It is shown that during interphase, Eg5 associates with ribosomes and is required for efficient protein synthesis.
The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.
PMCID: PMC3172266  PMID: 21795388
3.  The Role of Molecular Microtubule Motors and the Microtubule Cytoskeleton in Stress Granule Dynamics 
Stress granules (SGs) are cytoplasmic foci that appear in cells exposed to stress-induced translational inhibition. SGs function as a triage center, where mRNAs are sorted for storage, degradation, and translation reinitiation. The underlying mechanisms of SGs dynamics are still being characterized, although many key players have been identified. The main components of SGs are stalled 48S preinitiation complexes. To date, many other proteins have also been found to localize in SGs and are hypothesized to function in SG dynamics. Most recently, the microtubule cytoskeleton and associated motor proteins have been demonstrated to function in SG dynamics. In this paper, we will discuss current literature examining the function of microtubules and the molecular microtubule motors in SG assembly, coalescence, movement, composition, organization, and disassembly.
PMCID: PMC3132543  PMID: 21760798
4.  Deficiency in Myosin Light Chain Phosphorylation Causes Cytokinesis Failure and Multipolarity in Cancer Cells 
Oncogene  2010;29(29):4183-4193.
Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which play an important part of malignant transformation in the most recent models tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. We show here that cancer cells often failed at cytokinesis due to decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light chain phosphorylation was restored to normal levels by phosphatase knockdown multinucleation, and multipolar mitosis were both markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light chain kinase (MLCK) in nonmalignant cells can recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.
PMCID: PMC2911497  PMID: 20498637
cytokinesis; myosin light chain kinase; multinucleation; multipolar spindles; myosin phosphatase; myosin regulatory light chain
5.  The c-Myc Target Glycoprotein1bα Links Cytokinesis Failure to Oncogenic Signal Transduction Pathways in Cultured Human Cells 
PLoS ONE  2010;5(5):e10819.
An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbα), a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbα in a p53-deficient background. GpIbα was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbα.
PMCID: PMC2876040  PMID: 20520840
6.  Microtubule Binding and Disruption and Induction of Premature Senescence by Disorazole C1 
Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was previously found to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly due to its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization with an IC50 of 11.8 ± 0.4 μM and inhibited [3H]vinblastine binding uncompetitively with a Ki of 4.5 ± 0.6 μM. We also found uncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1 with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.
PMCID: PMC2649750  PMID: 19066338
7.  Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1 
BMC Chemical Biology  2010;10:1.
Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.
In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.
Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
PMCID: PMC2834648  PMID: 20181182
8.  Cik1 Targets the Minus-End Kinesin Depolymerase Kar3 to Microtubule Plus Ends 
Current biology : CB  2005;15(15):1420-1427.
Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth [1–7]. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides [8–11]. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy [8–11]. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s [12–18]. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1•tubulin complex formation as observed for MCAK [12, 13, 15, 16, 18]. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.
PMCID: PMC2386176  PMID: 16085496
9.  Cost and utilization of blood transfusion associated with spinal surgeries in the United States 
European Spine Journal  2006;16(3):353-363.
The purpose of this study was to examine factors associated with the utilization and cost of blood transfusion during and post-spinal fusion surgery. A retrospective, observational study of 42,029 inpatients undergoing spinal fusion surgery in United States hospitals participating in the PerspectiveTM Comparative Database for inpatient use was conducted. Descriptive analysis, logistic regression, and ordinary least squares (OLS) regression were used to describe the factors associated with the use and cost of allogeneic blood transfusion (ABT). Hospitalization costs were $18,690 (SD=14,159) per patient, erythropoietin costs were $85.25 (SD=3,691.66) per patient, and topical sealant costs were $414.34 (SD=1,020.06) per patient. Sub-analysis of ABT restricted to users revealed ABT costs ranged from $312.24 (SD=543.35) per patient with whole blood to $2,520 (SD=3,033.49) per patient with fresh frozen plasma. Patients that received hypotensive anesthesia (OR,1.61; 95% CI, 1.47–1.77), a volume expander (OR,1.95; 95% CI, 1.75–2.18), autologous blood (OR, 2.04; 95% CI, 1.71–2.42), or an erythropoietic agent (OR=1.64; 95% CI, 1.27–2.12) had a higher risk of ABT. Patients that received cell salvage had a lower risk of transfusion (OR=0.40; 95% CI, 0.32–0.50). Most blood avoidance techniques have low utilization or do not reduce the burden of transfusion associated with spinal fusion.
PMCID: PMC2200697  PMID: 16463198
Spinal fusion surgery; Burden of illness; Blood transfusion; Cost
10.  Slk19p Is a Centromere Protein That Functions to Stabilize Mitotic Spindles 
The Journal of Cell Biology  1999;146(2):415-426.
We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability. Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability. When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed. A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body. Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules.
PMCID: PMC3206577  PMID: 10427094
spindle; Saccharomyces cerevisiae; kinetochore; motor; yeast
11.  The Saccharomyces cerevisiae Kinesin-related Motor Kar3p Acts at Preanaphase Spindle Poles to Limit the Number and Length of Cytoplasmic Microtubules 
The Journal of Cell Biology  1997;137(2):417-431.
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.
PMCID: PMC2139775  PMID: 9128252
12.  Overview 
PMCID: PMC4193424  PMID: 25372889
13.  Overview 
PMCID: PMC4193355  PMID: 25372065
14.  ADY1, A Novel Gene Required for Prospore Membrane Formation at Selected Spindle Poles in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2001;12(9):2646-2659.
ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.
PMCID: PMC59701  PMID: 11553705
15.  RHEUMATOID ARTHRITIS—An Evaluation of Long-Term Treatment with Cortisone 
California Medicine  1954;80(5):369-374.
Fifty-six patients with rheumatoid arthritis were treated continuously with cortisone for periods ranging between 4 and 38 months, in daily doses of 15 to 100 mg. Concomitant therapy included periods of rest, physical therapy, and salicylates.
The incidence of subjective improvement exceeded that of objective improvement.
The incidence of objective improvement was higher in females; also, in those patients whose disease was in an early stage and of short duration at the time therapy was begun, and who required relatively smaller maintenance doses of cortisone.
Therapeutic results were not affected by the age of the patient or by the presence of spondylitis.
Despite precautions, the long-term administration of cortisone was, in some patients, productive of serious undesirable side-effects.
Although cortisone usually suppressed the symptoms and signs of rheumatoid arthritis, progression of the disease was frequently noted during its long-term administration.
PMCID: PMC1531762  PMID: 13150213

Results 1-15 (15)