Office: 843-792-7475
Lab: 843-792-1180
Fax: 843-792-8565
Email: spicer@musc.edu
BSB-512D

Research Interests

Regulation of mRNA stability of oncogenes, translational control of gene expression; protein:RNA interactions,
I. Post-transcriptional regulation of human bcl-2 expression
The molecular mechanisms that regulate apoptosis are not yet completely understood; however, multiple lines of evidence point to a role for Bcl-2 in regulating programmed cell death. In mammalian species, the Bcl-2 gene encodes a 29-kDa protein that predominantly resides in the outer mitochondrial membrane and nuclear envelope. Several studies have demonstrated that chemotherapeutic agents can alter Bcl-2 activity in a variety of cancer cells.  Based on our examination of the effects of taxol and okadaic acid on bcl-2 expression in the human leukemia cell line HL-60, we hypothesize that chemotherapeutic agents induce destabilization of bcl-2 mRNA by regulating the interactions of bcl-2 mRNA binding proteins with specific cis-elements in bcl-2 mRNA. Accordingly, our objectives are to identify the sequences or structures in bcl-2 mRNA that determine its sensitivity to chemotherapeutic agents such as taxol, and to identify and characterize the trans-acting factors that interact with these sequences.  Because of its anti-apoptotic activity, high Bcl-2 expression by malignant cells is potentially an obstacle to some forms of chemotherapeutic treatment. Our preliminary studies have shown that taxol-induced modulation of bcl-2 mRNA levels occurs via mRNA destabilization. This suggests that factors that influence bcl-2 mRNA stability may have significant therapeutic value. Currently, little is known about post-transcriptional regulation of bcl-2. Studies in our laboratory seek to address this deficiency in our knowledge and to provide new insights into this potentially important mechanism of controlling levels of the proto-oncoprotein Bcl-2.
II. T4 regA protein:
How proteins recognize nucleic acids is a question that is central to understanding many of the primary functions (e.g. DNA replication, transcription and mRNA processing) in both procaryotic and eucaryotic cells. Currently, our understanding of protein:RNA interactions lags behind that of protein:DNA interactions, in part because RNAs are more varied in their structure. The bacteriophage T4 regA protein is a unusual translational repressor that is an excellent model system for the study of protein recognition of RNA.  RegA protein is uniquely promiscuous in its recognition of RNA, in that it binds to 15 - 30 specific mRNAs and distinguished these targets from among 150 other mRNAs present in the phage-infected cell. Comparison of the sequence of twelve known regA-regulated mRNAs indicates they do not have a simple conserved nucleotide sequence nor apparent conserved secondary structures. Thus, how regA protein recognizes it specific target RNAs is largely unknown and is the subject of our research.  The crystal structure of regA protein has recently been solved. This accomplishment expands the opportunity to achieve significant insights into the mechanism of regA protein: RNA recognition and translational repression. Thus, current studies are focused on the introduction of single-site mutations into two potential RNA-binding domains of regA protein and functional characterization of the mutant proteins. Mutations are also being introduced into synthetic RNAs corresponding to in vivo targets, to probe the location of contacts between protein side chains and RNA functional groups in the formation of specific complexes. Earlier studies focused on defining the recognition site in T4 gene 44, shown below. Future efforts will be directed at defining the critical regions in other mRNAs, to determine the characteristics common to two or more target in mRNAs.

Selected Publications

• Otake, Y., Soundararajan, S., Sengupta, T.K., Kio, E.A., Smith, J.C., Pineda-Roman, M., Stuart, R.K., Spicer, E.K. and Fernandes, D.J. “Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl-2 mRNA". Blood, 109: 3069-3075, (2007).
• Bose, S.K., Sengupta, T.K., Bandyopadhyay, S. and Spicer, E.K. “Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP complexes". Biochem. J., 396: 99-107, (2006)
• Otake, Y., Sengupta, T.K., Bandyopadhyay, S., Spicer, E.K., and Fernandes, D.J. (2005) Retinoid-Induced Apoptosis in HL-60 cells is Associated with Decreased Stabilization of bcl-2 mRNA by Nucleolin.  Molecular Pharmacology, 67, 319 -326
• Yang, W., White, B., Spicer, E.K., and Hildebrandt, J. (2004) Complex haplotype structure of the human GNAS gene identifies a recombination hot spot centered on a SNP associated with hypertension. Pharmacogenetics 14: 1-7.
• Otake, Y., Sengupta, T.K., Bandyopadhyay, S., Spicer, E.K. and Fernandes, D.J. (2004) Drug-induced destabilization of Bcl-2 mRNA: A new Approach for Inducing Apoptosis in Tumor Cells.  Current Opinions in Investigational Drugs 5(6): 616-622.
• Sengupta, T.K,., Bandyopadhyay, S., Fernandes, D. and Spicer, E.K. (2004) Identification of nucleolin as an AU-rich element binding protein involved in Bcl-2 mRNA stabilization. J. Biological Chemistry 279: 10855-10863.
• Bandyopadhyay, S., Sengupta, T.K., Fernandes, D. and Spicer, E.K. (2003) Taxol- and okadaic acid-induced destabilization of bcl-2 mRNA is associated with decreased binding of proteins to a bcl-2 instability element. Biochemical Pharmacology, 66: 1151-1162.
• Ullian, M.E., Gantt, B.J., Ford, A.K., Tholanikunnel, B.G., Spicer, E.K. and Fitzgibbon, W.R. (2003) Potential importance of glomerular citrate synthase activity in remant nephropathy. Kidney Int., 63(1): 156-164.
• Sengupta, T.K., Gordon, J., and Spicer, E.K. (2001) RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities. Nucleic Acids Research, 29(5): 1175-1184.
• Gordon, J., Sengupta, T.K., Phillips, C.A., O'Malley, S.M., Williams, K.R., and Spicer, E.K. (1999) Identification of the RNA Binding Domain of T4 RegA Protein by Structure-based Mutagenesis. J. Biological Chemistry 274(45): 32265-32273.
• Goodrich, L.D., Lin, T-C., Spicer, E.K., Jones, C., and Konigsber, W.J. (1997) Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins. Biochemistry 36(34): 10474-10481.
• Narasimhan, S., Armstrong, M., McClung, J.K., Richards, F.R., and Spicer, E.K. (1997) Prohibitin, a putative negative control element present in Pneumocystis carinii. J. Infection and Immunity 65(12): 5125-5130.
• Phillips, C.A., Gordon, J., and Spicer, E.K. (1996) Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions. Nucleic Acids Research 24: 4319-4326.