Probing Mechanical Properties of Jurkat Cells under the Effect of ART Using Oscillating Optical Tweezers

Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effect of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an oscillating optical tweezer was employed to characterize the frequency-dependent mechanical properties of Jurkat cells exposed to the chemotherapeutic agent, artesunate (ART). A motion equation for a bead bound to a cell was applied to describe the mechanical characteristics of the cell cytoskeleton. By comparing between the modeling results and experimental results from the optical tweezer, the stiffness and viscosity of the Jurkat cells before and after the ART treatment were obtained. The results demonstrate a weak power-law dependency of cell stiffness with frequency. Furthermore, the stiffness and viscosity were increased after the treatment. Therefore, the cytoskeleton cell stiffness as the well as power-law coefficient can provide a useful insight into the chemo-mechanical relationship of drug treated cancer cells and may serve as another tool for evaluating therapeutic performance quantitatively

Molecular cloning and characterization of the 5'-flanking and promoter region of the human dopamine D5 receptor gene

grantor: University of TorontoGenomic and overlapping cDNA clones encompassing the entire 5\sp\prime-untranslated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized intron (of either 179 or 155 bp). In this thesis it was demonstrated that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5\sp\prime to the transcription-initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Sp1 and AP1. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5\sp\prime of the transcription initiation site were able to stimulate transcription in SK-N-SH and GH3 cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained positive modulators between nucleotides $-$52 to $-$119 and $-$120 to $-$182 upstream from the site of transcription initiation. Furthermore, a negative modulator $-$251 to $-$500 nucleotides upstream from the site of transcription initiation was also indicated. Within the positive modulatory region there existed a small dinucleotide repeat, termed (TC)\sb{13}. Genomic DNAs from 18 unrelated schizophrenic (n = 7), Parkinson's diseased (n = 2), Huntington's diseased (n = 6) and normal healthy individuals (n = 3) were analysed using a single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed the existence of two additional alleles, termed (TC)\sb{12} and (TC)\sb{14}. Neither form significantly altered D5 promoter-mediated luciferase activity when compared to that of the wild-type control, suggesting that differences in the number of dinucleotide repeats are not likely of any functional consequence for D5 transactivation. Proteins from GH3 and SK-N-SH cell nuclear extracts were able to bind oligonucleotide probes directed to the positive and negative modulator regions of the D5 promoter in electrophoretic mobility shift experiments. Neither of the Sp1 consensus sequences appeared to bind to nuclear proteins and the involvement of Pit-1 and AP1 are currently under investigation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridisation analysis of monkey and human brain using a 5\sp\prime DS-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labelled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor mediated events.Ph.D

Four different statistical models to analyze cell parameters changes by changing ART dosages comparing to control group.

<p><i>X</i>₀: the displacement of the bead on the cell membrane caused by the fluidity of the cell, <i>v</i>: the degree of non-linearity of the cell’s material, <i>η</i>_<i>cell</i>: cell’s viscosity coefficient</p><p>Four different statistical models to analyze cell parameters changes by changing ART dosages comparing to control group.</p

Estimated cell parameters under the control condition and four different dosages of ART.

<p><i>X</i>₀: the displacement of the bead on the cell membrane caused by the fluidity of the cell, <i>v</i>: the degree of non-linearity of the cell’s material, <i>η</i>_<i>cell</i>: cell’s viscosity coefficient</p><p>Estimated cell parameters under the control condition and four different dosages of ART.</p

Epigenetic Characterization of the Growth Hormone Gene Identifies SmcHD1 as a Regulator of Autosomal Gene Clusters

Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin β cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS &amp; SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes

Sample immunofluorescnet images of the Jurkat cells (left: cells exposed to 50 μg/ml ART, right: non-treated cells).

<p>Sample immunofluorescnet images of the Jurkat cells (left: cells exposed to 50 μg/ml ART, right: non-treated cells).</p

Stiffness versus frequency under the non-treated condition for frequencies between 0.1~100 Hz.

<p>Each data point represents the median value of 20 cells. <i>Y</i> increases with increasing frequency. Since the axes are logarithmic, the power-law dependency appears as a straight line with a slope of <i>x</i>—1. The power-law exponent is estimated to be 0.34, which shows a weak power-law dependency of <i>Y</i> on frequency.</p

Sample cell-bead complex image with R-contact shown.

<p>The contact radius was determined by fitting a circle to the cell outline and another to the bead outline in the experimental images. A cord was drawn where the two circles overlapped. The distance from the bead circumference to the cord was defined as the contact radius.</p