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RISE Students' Research Abstracts / 2009 - 2010

THE POTENTIAL OF LACTOBACTILLUS AS A CARRIER OF CHLAMYDIA TRACHOMATIS CRYPTIC PLASMID ANTIGENS IN CHLAMYDIA VACCINE DEVELOPMENT. A. Campbell1, E. Ekong4, G. Ifere1, K. Joseph4, T. Belay3, F. Eko2, N. Diala1, Q. He2, C. Black4, J. Igietseme4, and G. Ananaba1. 1Clark Atlanta University, Atlanta, GA, 2Morehouse School of Medicine, Atlanta, GA, 3 Bluefield State University, Bluefield, WV, 4Centers for Disease Control and Prevention, Atlanta, GA.

Chlamydia trachomatis genital infection is a prevalent bacterial sexually transmitted disease that can have serious consequences such as infertility, ectopic pregnancy, and pelvic inflammatory disease if it goes untreated.  Current vaccinology strategies are attempting to produce an effective vaccine that would confer immunity against genital chlamydial infection and eliminate chlamydial pathology.  Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function.  The Chlamydia cryptic plasmid (pCT) encodes eight putative open reading frames (ORFs), designated pORF1 to -8.  pORF 5 encodes a 28-kDa protein, pgp3, with unknown function.  The goal is to elucidate the role of CT cryptic plasmid in pathogenesis of Chlamydia by identifying immunopathogenic cryptic plasmid antigens that drive Chlamydia pathology.   We hypothesize that cryptic plasmid antigens maybe used in the development of an effacious vaccine against Chlamydial genital infection.  We used a genetic approach to assess the role of the functional genes in CT cryptic plasmid in the pathogenesis of infertility.  Lactobacillus, a commensal bacterium of the human gastrointestinal flora, was used as a surrogate vehicle to express CT plasmid genes in vivo.  In our laboratory, we have evaluated the effect of Chlamydia cryptic plasmid in fertility, and found that plasmid deficient Chlamydia does not cause pathology.  Additionally, we have isolated the eight open reading frames, and developed a live recombinant vaccine scheme that utilizes Lactobacillus fornicalis as a live delivery vehicle of CT cryptic plasmid pgp3.  A vaccine encompassing live recombinant Lactobacillus with CT cryptic plasmid antigen may possibly induce a strong cell-mediated and humoral immune response in a mouse model protecting against Chlamydia trachomatis challenge and pathology.  Our vaccine scheme can be used for general application of vaccinology efforts towards other diseases such as prostate cancer. 

Acknowledgements:  Supported by NIH grants GM08247 and A141231.

Angela Campbell's Portfolio


DOWN REGULATION OF PTEN IS OBSERVED IN CXCR4 EXPRESSING PROSTATE CANCER CELLS. Mahandranauth A. Chetram, Cimona V. Hinton, Clark Atlanta University, Atlanta, GA.

The progression of human prostate cancer is a result of metastasis from the primary tumor to vital organs. Metastasis is a complex process that involves invasion, intravasation, extravasation, and metastatic colonization. To date, the biology underlying the various mechanisms of metastasis has not been elucidated. Chemokines are pro-inflammatory molecules that bind to chemokine receptors, which are G-protein coupled receptors (GPCR). The interaction between chemokines and their receptors results in a diverse array of biological and biochemical functions, such as chemotaxis, hematopoiesis and angiogenesis. Likewise, neoplastic cells employ chemokines and their receptors to promote metastasis and encourage cell survival. In prostate cancer, the CXCR4 chemokine receptor is overexpressed on the cell surface, which directs metastatic cells to tissues where its ligand is overexpressed, such as the bones and lungs. The Phosphate and Tensin homolog deleted on chromosome 10 (PTEN) is the second most mutated tumor suppressor in human cancer and has been shown to be inactivated in metastatic prostate cancer cells. Furthermore, Gao et al showed that reconstituted PTEN in Jurkat T-cells down-regulated CXCR4-mediated chemotaxis. Therefore, we hypothesize that the absence of PTEN expression correlates with an upregulation of CXCR4-mediated functions in prostate cancer cells. We analyzed the expression of CXCR4 by flow cytometry and observed that CXCR4 was highly expressed in human metastatic prostate cancer cell lines, PC3 and LnCaP. PTEN expression was analyzed by western blot and RT-PCR. We did not observe expression of PTEN in PC-3 and LnCaP cells at the protein level. However, expression at the mRNA level was observed in LnCaP cells. In summary, our results showed that CXCR4 is over expressed in PTEN-null prostate cancer cell lines. In future studies, we will investigate if the decrease expression of functional PTEN promotes CXCR4-mediated metastasis in prostate cancer cells.

Acknowledgements: RISE program and RCIM grant number 2G12RR003062-22.

Mahandranauth A. Chetram's Portfolio



Fabrication of Functional Conductive Nanofibers composed of α,ω-bi-DNP-poly(2-methoxystyrene), polystyrene and single walled carbon nanotubes for studying material-cell interactions. Omotunde Olubi
1, Deepti Gadi2, Biswajit Sannigrahi1, Maher Atteya3, M.D. Williams4,Barbara Baird2, Ishrat Khan1; 1Department of Chemistry, Clark Atlanta University, Atlanta, GA; 2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY; 3Department of Chemistry, Georgia Perimeter College, Clarkston, GA; 4Department of Physics, Clark Atlanta University, Atlanta, GA.

Conductive fibers decorated with α,ω-bi-DNP (dinitrophenyl) groups capable of specifically binding with anti-DNP IgE have been prepared by electrospinning a solution of α,ω-bi-DNP-poly(2-methoxystyrene), polystyrene and single walled carbon nanotubes (SWCNT). The nanowires (200nm) were electrospun onto a silicon wafer substrate at a voltage of 10kV using either DMF or chlorobenzene.  The bi-nitrophenyl (DNP)- functional groups were tethered to the fibers via oligo(oxyethylene) spacers. The α,ω-bi-DNP-poly(2-methoxystyrene) functional polymers were synthesized in the lab by first preparing the α,ω-di-hydroxyl-poly(2-methoxystyrene) by living anionic polymerization, followed by the functionalization of the di-hydroxyl polymer with the DNP functional group. The functional polymers have been characterized by 1H and 13C NMR, FT-IR, DSC and GPC. Furthermore, we have also prepared conductive (based on polypyrroles) nanowire structures decorated with functional (DNP) groups capable of specifically engaging target anti- DNP IgE and IgE on mast cell surfaces. The binding affinity of nanowires containing 1% single-walled Carbon Nanotubes with anti-DNP IgE has been studied via fluorescently labeled FITC-IgE. The success achieved so far in this investigation suggests the possibility of developing functional nanofibers as the active component in biosensors.

Acknowledgements:  These studies were supported by the MBRS RISE Program-NIH/NIGMS Grant #5R25GM060414.

Omotunde Olubi-Oni's Portfolio


SNAIL 1 REGULATES MIGRATION THROUGH Rac-1 AND INVASION THROUGH uPA-uPAR SIGNALING IN PROSTATE CANCER CELLS. Diandra D. Randle, Shineka Clarke, Danielle McKeithen and Valerie Odero-Marah, Cancer Center for Research and Therapeutic  Development, Department of Biological Sciences, Clark Atlanta University, Atlanta, Georgia.

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -b (TGF-b) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by Rac1 GTPase, urokinase (uPA) and its receptor (uPAR) activities. Rac1, a Rho GTPase, when converted from the inactive Rac1-GDP to the active Rac1-GTP form localizes at lamellipodia to stimulate cell movement. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including migration and invasion. 22Rv1, LNCaP and ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail which led to increased cell migration and invasion. The levels of Snail, Rac1, uPA, and uPAR were measured by Western Blot analysis.  Rac1 activity was compared between Neo- and Snail-transfected cells by immunoprecipitating Rac1- GTP followed by western blot analysis with anti-Rac antibody. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. The data suggests that overexpression of Snail1 increased Rac1 activity in ARCaP and LNCaP cells. Snail overexpression in LNCaP cells increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from ARCaP, LNCaP and 22Rv1 cells. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of Rac and uPA activities.

Acknowledgements: These studies were supported by the NIH/NCRR/RCMI grant G12RR03062 and NIH P20 grant 1P20MD002285.


Mesoporous Materials for the Absorption of Proteins. Kyarra Thompson and Conrad Ingram, Department of Chemistry, Clark Atlanta University, Atlanta, GA  30314.

Mesoporous silicates are potentially effective for the absorption of proteins by entrapment or immobilization in their organic and inorganic-organic hybrid structures.  This phenomenon is potentially useful for protein separation, production of biosensors and biocatalysts, and for drug delivery applications.  Previous researchers have attempted to absorb whole proteins in the void volume of mesoporous silicates such as MCM-41 (pore size, 2 nm) and SBA (pore size 6 nm).  Our research is focused on chemical modification of surface MCM-41 and SBA-15 and to enhance their adsorption capacity and selectivity for proteins into their mesoporous structures. The absorption kinetics and isotherms are being studied as a function of the materials’ pore size, surface area, pore chemistry, solution pH, and protein structures.

Acknowledgements:  These studies were supported by the MBRS RISE Program-NIH/NIGMS Grant #5R25GM060414.

Kyarra Thompson's Portfolio



ROLE OF NODAL IN PROSTATE CANCER PROGRESSION. BaoHan T. Vo
and Shafiq A. Khan. Department of Biological Sciences and Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, Georgia 30314.

Nodal is an embryonic morphogen belonging to the TGF-β superfamily that has been demonstrated to inhibit differentiation and to maintain the pluripotency of human embryonic stem cells. Like other members of the TGFβ family, Nodal signals through type I and type II serine/theorine kinase receptor complexes and intracellular Smad proteins which translocate to the nucleus where they regulate gene expression. In addition, Cripto, a member of the epidermal growth factor–Cripto-FRL-Criptic (EGF-CFC) protein family, has also been implicated in Nodal signaling. So far, Nodal has been studied in breast, ovarian, and melanoma cancer cell lines; however, very little is known about Nodal in prostate cancer progression. Therefore, the purpose of this study was to determine Nodal expression and its biological effects in human prostate cell lines. RT-PCR and Western blot analysis were performed to analyze the expression of Nodal and Nodal receptors and its effect phosphorylation of Smad 2/3 in prostate cell lines. Effects of human recombinant Nodal (rhNodal) on proliferation of DU145, LNCaP, PC3, and WPE cells were determined by MTT assay and 3H-Thymidine incorporation assay. We observed that Nodal protein is highly expressed in DU145, LNCaP-C81, and WPE (stem) cells and weakly expressed in RWPE1 and RWPE2 cells; however, Nodal protein expression was undetectable in PC3 and PC3M cells. Nodal receptor mRNAs were expressed, at varying levels, in prostate cell lines. Treatment with exogenous, Nodal caused a dose-dependent inhibition of proliferation in DU145 and WPE cells, but Nodal had no effects on the proliferation of LNCaP and PC3 cells. These findings demonstrate that Nodal is expressed in some prostate cancer cell lines and prostate stem cells. Nodal induced a dose and time dependent phosphorylation of Smad 2/3 in DU145 cells. In conclusion, we demonstrated for the first time that Nodal is expressed in prostate cancer cell lines and it inhibits proliferation of DU145 cells and WPE (stem) cells. These effects may be mediated via a Smad 2/3 dependent mechanism.

Acknowledgments: These studies were supported by the NIH/NCRR/RCMI grant  #2G12RR003062, NIH P20 grant #5P20MD002285-02, DOD grant # W8I-08-1-0077 and NIGMS/NIH grant #5R25GM0604.

BaoHan T. Vo's Portfolio



INHIBITION OF CXCR4-MEDIATED PROSTATE BONE METASTASIS BY THE TUMOR SUPPRESSOR PTEN. T’Shane C. Williams, Amber L.Taylor, Mahandranauth A. Chetram and Cimona V. Hinton, Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, Georgia.

Bone is the most common site for advanced metastatic prostate cancers. Once in the bone, tumors are incurable to therapy, and contribute to increased mortality through unknown mechanisms. The tumor and bone interact in a vicious cycle, where tumor-secreted factors stimulate bone cells, which in turn release growth factors and cytokines that act on tumor cells to further influence metastasis.  Metastasis is a complex, multistep process; however, its mechanisms have not been fully elucidated.  Activation of chemokine receptors results in a diverse array of biological functions, such as hematopoiesis, angiogenesis and metastasis. In our prostate cancer model system, CXCR4 is overexpressed on the cell surface, which directs metastatic cells to tissues, where its ligand is overexpressed. Additionally, prostate cancer cells have been shown to exhibit a loss of the tumor suppressor PTEN, which also correlates with increased malignancy.  Our aim was to determine whether reconstituting PTEN in CXCR4-overexpressing prostate cancer cells inhibited their ability to migrate through bone marrow endothelial cells. We analyzed an array of human prostate cancer and human embryonic kidney cancer cells (control) for PTEN expression by Western blot analysis and observed that PTEN was not expressed in prostate cancer cell lines that represented an advanced, metastatic stage.   We also analyzed the expression of CXCR4 by flow cytometry and observed that CXCR4 was highly expressed in human metastatic prostate cancer cells. We reconstituted PTEN into prostate cancer cells by transfection, and analyzed their migration through human bone marrow endothelial cells to their ligand, SDF1α.  We observed that upon transfection of PTEN, the migratory behavior of PC3 cells was altered, thus inhibiting metastasis.   In future studies, we will determine whether antagonists of CXCR4 and siRNA against AKT will inhibit prostate bone metastasis.

Acknowledgements:  These studies were supported by the NIH/NCRR/RCMI grant #2612RR003062 and MBRS RISE Program NIH/NIGMS Grant #5R25GM060414.

T’Shane Candice Williams' Portfolio