91制片厂

My laboratory has had a long-standing interest in the regulation of gene expression and cell proliferation as they relate to cancer. Our work has focused on the transcriptional regulation of genes that lack a TATA element in their promoter, which was once thought to be a canonical feature. Genes regulated by these so-called TATA-less promoters include genes involved in regulation of many metabolic processes, DNA replication, DNA repair, and apoptosis, as well as growth factors and their receptors, oncogenes and tumor suppressors. Promoters for these genes are GC-rich and most contain multiple sites that bind the transcription factor Sp1.

We and others demonstrated that in TATA-less promoters, Sp1 functions to control transcription initiation by recruiting the general transcription machinery and co –activators (and repressors) which modify local chromatin structure.  Although considered a "general" transcription factor, regulation of transcription of a large number of genes in response to a wide array of signals has been ascribed to Sp1. Sp1 activity is regulated by post-translational modifications, including phosphorylation, acetylation, O-linked glycosylation, sumoylation, ubiquitylation, and methylation. These modifications affect not only DNA binding, but also Sp1 activity and interactions with other factors. Our work has largely focused on regulation of Sp1 activity through modulation of phosphorylation, with some work and significant interests related to acetylation, sumoylation and glycosylation.

Several years ago, a graduate student in the lab discovered that Sp1 is significantly phosphorylated in response to DNA damage. Sp1 is phosphorylated by several different kinases at many different sites to modulate its activity in response to various signals. Much of our current work is focused on phosphorylation in response to DNA damage and the role of Sp1 in the cellular response to damage. Eleven of the 96 Ser residues in Sp1 are SQ sequences clustered in the glutamine-rich transactivation domains; S/TQ cluster domains (SCDs) are characteristic of proteins phosphorylated by ATM/ATR in response to DNA damage.  We have found that Sp1 is phosphorylated by ATM on several Ser residues in response to DNA damage and that its phosphorylation is involved in the increased sensitivity to DNA damage observed in cells depleted of Sp1. Phosphorylation on S101 is required for additional phosphorylation, i.e. pS101  primes for additional phosphorylation. We have shown by immunofluorescence/confocal microscopy and chromatin immunoprecipitation that pSp1-S101  is localized in close proximity to DNA double strand breaks.  Sp1 is involved in nucleosome displacement at break sites and facilitates DNA repair. 

Sp1 is overexpressed in many tumors and is a bad prognostic indicator.  In normal cells, Sp1 overexpression drives cells into apoptosis.  We have also studied  the role of Sp1 in the induction of apoptosis after DNA damage. At high levels of damage, Sp1 is specifically cleaved at D183 by caspases and blocking caspase-mediated cleavage (D83A) protects cells from apoptosis indicating that Sp1 plays a direct role in promoting apoptosis. Cleavage at D183 releases a 70kDa fragment which has nearly 100% transcription activity.  We propose that Sp1-70 may transcriptionally activate pro-apoptotic genes and /or inhibit survival genes, many of which have Sp1 binding sites and we are currently testing this hypothesis.

Additional Projects Under Way

Defining the role of activation of the DNA damage response (DDR) in herpes simplex virus (HSV-1) infection.  Many viruses activate the cellular DNA damage response during viral infection.  The mechanisms by which this occurs are not understood. We are using epithelial cells from the cornea, oral cavity and esophagus and seek to determine the upstream events that activate ATM early in infection and the mechanistic consequences of ATM activation in viral replication.  Pharmacologic or RNAi targeting  ATM or Chk2 early in infection completely blocks HSV-1 production. 

Exploring the role of Sp1 in diabetic retinopathy. Diabetic retinopathy is a very early manifestation of diabetes and a leading cause of blindness in the developed world.  It is characterized by weak and leaky retinal microvasculature, followed by the overgrowth of fragile new vessels. The proangiogenic factor VEGF-A (vascular endothelial growth factor A) contributes significantly to retinal neovascularization.  Many of the transcription factors that regulate VEGF-A, notably Sp1 are modified by O-linked N-acetylglucosamine (O-GlcNAc) to modulate their activity. Elevated glucose results in increased overall O-GlcNAcylation of proteins. Addition and removal of O-GlcNAc to Ser and Thr residues is catalyzed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Using rat retinal capillary endothelial cells and human retinal pigment epithelial cells to assess the importance of OGT in glucose-regulated VEGF-A production, we have found that VEGF-A transcript and protein levels are increased by high glucose, and that shRNA-mediated depletion of OGT abrogates the increased VEGF-A induced by hyperglycemia. Pharmacologic inhibition of OGA, which elevates O-GlcNAcylation, increases VEGF-A, implicating OGT as a critical regulator of VEGF-A. Further experiments involve characterization of the VEGF promoter and identification of the sites of O-GlcNAcylation on Sp1 and other transcription factors.

Characterizing the effects of non-thermal plasma on mammalian cells.  In collaboration with Gary Friedman and Alex Fridman (College of Engineering), we are studying the effects of non-thermal plasma on cells. Plasma comprises electrically charged molecules, electrons as well as some highly active neutral molecules (electronically excited atoms and radicals) that can be produced through application of a strong electric field. In this work we employ electrodes with a dielectric barrier to produce plasma whose average temperature is close to room temperature. We have shown that when applied to a solution, non-thermal plasma produces stable  reactive oxygen species in a dose-dependent and highly controllable manner. The goal is to characterize the type of DNA damage induced by plasma and to determine its effects on virus replication and to develop cold plasma for sterilization of surfaces, particularly wounds, and to induce apoptosis in cancer cells by local administration. We are characterizing the reactive oxygen species that are produced and their effects on DNA.

"Third Annual Medical Student Research Day"
College of Medicine Newsroom (March 21, 2014)

Torabi B, Flashner S, Beishline K, Sowash A, Donovan K, Bassett G, Azizkhan-Clifford J
Apoptosis; 23(1):65-78. doi: 10.1007/s10495-017-1437-4, 2018 PMID:29236199, Jan 2018

Beishline K, Azizkhan-Clifford J
Methods Mol Biol.; 1170:41-59. doi: 10.1007/978-1-4939-0888-2_3, PMID:24906308 (2014)

Alekseev O, Donovan K, Limonnik V, Azizkhan-Clifford J
Transl Vis Sci Technol.; 3(2):2. eCollection 2014 Mar PMID:24757592 (March 27, 2014)

Alekseev O, Donovan K, Azizkhan-Clifford J
Invest Ophthalmol Vis Sci.; 55(2):706-15. doi: 10.1167/iovs.13-13461, PMID:24370835 (February 3, 2014)

"ErbB2, FoxM1, and 14-3-3ζ Prime Breast Cancer Cells for Invasion in Response to Ionizing Radiation"
D Kambaugh, V Soto, P Lelkes, J Azizkhan-Clifford*, and M Reginato*
Oncogene advance online publication; doi: 10.1038/onc.2012.629 (January 14, 2013)
*Co-corresponding authors

Alekseev O, Tran AH, Azizkhan-Clifford J
J Vis Exp; (69):e3631. doi: 10.3791/3631, PMID:23149439 (November 3, 2012)

Beishline K, Kelly CM, Olofsson BA, Koduri S, Emrich J, Greenberg RA, Azizkhan-Clifford J
Mol Cell Biol.; 32(18):3790-9. doi: 10.1128/MCB.00049-12. (Epub 2012 Jul 23) PMID:22826432 (September 2012)

"Effects of Non-Thermal Plasma on Mammalian Cells"
S Kalghatgi, CM Kelly, E Cerchar, B Torabi, O Alekseev, A Fridman, G Friedman and J Azizkhan-Clifford
PLOS One, 21;6(1):e162702010 (2011)

Astrinidis A, Kim J, Kelly CM, Olofsson BA, Torabi B, Sorokina EM, Azizkhan-Clifford J
Genes Chromosomes Cancer; 49(3):282-97. doi: 10.1002/gcc.20739, PMID: 20013896 (March 2010)

"Phosphorylation of Sp1 in Response to DNA Damage by Ataxia Telangiectasia-Mutated Kinase"
B Olofsson, CM Kelly, J Kim, S Hornsby and J Azizkhan-Clifford
Molecular Cancer Research, 5(12):1319-1330 (2007)

"Dual regulation of the anaphase promoting complex in human cells by cyclin A-Cdk2 and cyclin A-Cdk1 complexes"
J Mitra, GH Enders, J Azizkhan-Clifford and KL Lengel
Cell Cycle, Mar;5(6):661-6 (2006)