Postdoctoral Training and Faculty Appointments:
Since graduating from UT in 1994, Michael Carastro’s research interests have focused on the molecular etiology of human cancers, including the molecular mechanisms that maintain cellular DNA integrity and regulate gene expression. His early research work was in DNA replication/repair, but his dissertation research serendipitously progressed into a novel area of gene expression regulation, called nonsense-mediated mRNA decay (NMD). NMD is a translation-dependent mRNA degradation process that only targets mRNAs containing a translation termination codon recognized as “premature.” Carastro was primary author of a 2002 Nucleic Acids Research article that first reported a connection between cellular DNA replication (DNA polymerase delta) and the nonsense-mediated mRNA decay (NMD) helicase (HUPF1, helicase required for NMD). Since the initial discovery, other groups have published results following this pioneering work &/or cited it in review articles, including Current Opinion in Genetics and Development (Culbertson & Leeds, 2003), Nature Reviews Molecular Cell Biology (Maquat 2004), Molecular Microbiology (de Pinto et al., 2004), Journal of Cell Science (Maquat 2005), Molecular and Cellular Biology (Whittmann et al, 2006), Current Biology (Azzalin & Lingner, 2006) and Cell Cycle (Azzalin & Lingner, 2006).
Carastro’s most recent research articles report on novel molecular mechanisms of gene expression involving tumor suppressor genes, including p53 and prohibitin (another important tumor suppressor gene). Some recent research in Carastro’s laboratory is focused on defining the role of putative uORFs in the mRNA 5’-leaders of forkhead transcription factor (FOXO3a, a bona fide tumor suppressor gene) and retinoic acid receptor-α (involved in cell growth). Finally, he has also taught, written, and/or carried out research in the areas of biotechnology in biomedical sciences, pharmacogenetics/genomics, and nutriceuticals in cancer prevention/treatment.
Current Research Projects
Deleterious changes in cellular DNA are the hallmark of cancer. Damaged DNA leads to the changes observed in cancerous cells, like uncontrolled growth and the ability to relocate in the body (metastasize). Human cells possess multiple mechanisms that repair DNA damage and other cellular mechanisms that eliminate severely damage cells through an orderly "cellular suicide" process, known as apoptosis. Tumor suppressor genes are responsible for protecting human genomic DNA from damage by "turning on" genes that: (1) are required for DNA repair, and/or (2) activate/execute cellular apoptosis. This dual function restricts damaged (and potentially cancerous) cells from growing uncontrollably. If an individual's DNA has deleterious mutations in tumor suppressor genes (especially p53), then that individual will have reduced/null tumor suppressor function and is much more likely to develop a variety of cancers.
It has been known for decades that p53 is mutated in most cancers. Tens of thousands of research articles underscore the fact that p53 is a tumor suppressor gene with a critical role in the control of cell-cycle progression, apoptosis, and maintenance of genomic stability. In fact, p53 is so important that it has been hailed as "The Guardian of the Genome." However, our understanding of the mechanisms that regulate p53 gene expression is still incomplete. It is known that DNA damage and some cellular stresses induce p53 transcription. Further, the stability and function of p53 protein is post-translationally regulated by various post-translational modifications. However, cellular p53 protein concentration is also a major determinant of function. Therefore, the regulation of p53 mRNA translation could potentially impact on p53 function.
Since the protein levels of p53 are important, Carastro engaged in research designed to probe the translation of p53 mRNA into p53 protein. In a 2003 Molecular Cancer Research article co-authored by Carastro, it was reported that two forms of p53 mRNA are differentially expressed in normal versus tumor tissue/tumor-derived cell lines (Strudwick et al, 2003). Also, it was reported that the p53 mRNA species with the longest leader sequence (p53 mRNA-L, initiated from the P0 transcription start site) was detected only in normal tissues, but the shortest species of the p53 message (p53 mRNA-S, initiated from the P1 transcription start sites) was detected primarily in tumor tissue/tumor-derived cell lines [See p53 gene loci, p53 mRNA-L, & p53 5’-leaders figure]. Furthermore, the p53 mRNA-L 5’-leader sequence was reported to impose translational repression, but shorter p53 mRNA 5-leader sequences did not repress translation. Therefore, it was suggested that normal cells primarily express the translationally repressed p53 mRNA-L species (makes less p53 protein), but tumor-derived cells primarily express p53 mRNA-S that is not translationally repressed (makes more p53 protein).
Recent unpublished work on the continuation of this p53 project confirms that the uORF in the p53 mRNA-L 5'-leader is translatable and that uORF translation triggers preferential destabilization of p53 mRNA-L. Experimental data from analyses of human p53 mRNA 5'-leader sequence is consistent with (i (1)) repressed translation of a reporter ORF in cis, (ii (2)) the p53uORF23 peptide sequence is translatable in human cells, and (iii (3)) the uORF-containing p53 mRNA-L sequence selectively destabilizes mRNA by a translation- and kinase-dependent mechanism. Firstly, elimination of the p53 uORF relieves translational repression. Secondly, quantitative Real Time-PCR analyses of endogenous p53 mRNAs from human cells indicate that p53 mRNA-L is selectively increased when translation or kinase activity are chemically inhibited. Collectively, these data are consistent with (i (1)) p53 uORF-translation induced degradation of p53mRNA-L (likely by NMD), and (ii (2)) p53 mRNA-L uORF-dependent translational repression.
Carastro's current hypothesis is that p53 mRNA-L is a polycistron, which encodes p53 protein and p53uORF23. Further, the p53uORF23 peptide functions as a cyclin-dependent kinase inhibitor. Lastly, only p53 mRNA-L is a degraded by uORF translation-dependent NMD. [See Differential p53 mRNA expression model figure]
Student Research Projects
The molecular mechanisms regulating tumor suppressor genes (p53 and forkhead) and a cellular growth control gene (retinoic acid receptor-α) that are being studied in Carastro's laboratory involve (1) the translational repression imposed on messenger RNAs (mRNAs) by an upstream open reading frame in the mRNA 5'-leader sequence, and (2) a mRNA degradation process, called non-sense mediated mRNA decay (NMD), triggered by some uORF-translation.
Research project available for undergraduate participation include:
- The p53uORF23 peptide will be expressed under the control of an inducible promoter in human cells and/or a transgenic mouse model in order to more precisely define a role for p53uORF23 peptide in cellular physiology. Therefore the goal of this aim is to discern any changes in cell morphology, proliferation rate, and/or apoptosis frequency associated with expression of the p53uORF23 peptide.
- Determine whether the peptide encoded by the uORF in p53 mRNA-L has the ability to: (1) bind to any cellular cyclins via direct protein-protein interactions, or (2) serve as a substrate for phosphorylation by a cellular kinase. The goals of this project are to demonstrate (1) that the putative cyclin-binding motif in p53uORF23 confers specificity to cellular cyclin(s), and (2) that the putative phosphorylation motif within p53uORF23 can serve as a cellular kinase substrate.
- Assay for the presence of p53uORF23 peptide by screening numerous samples of cell lines, tumors and normal tissues (using LC-MS/MS analysis techniques). The goal of this project is to demonstrate the presence of endogenous p53uORF23 peptide in non-cancerous human cells/tissues exclusively.
- Further define the uORF-dependent p53 mRNA-L destabilization mechanism as nonsense-mediated mRNA decay (NMD), using both molecular and genetic techniques. The goal of this project is to demonstrate that an intact uORF in p53 mRNA-L is required and sufficient to elicit NMD-mediated degradation.
- Engineer and generate mutant vectors for uORF-containing 5'-leader sequences of forkhead and retinoic acid receptor-α (RXRa) by using site-directed mutagenesis. Assay for translational repression and uORF-translation induced NMD. The goal of this project is to define the role of uORFs in forkhead and RXRa.
Peer-Reviewed Research Articles
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. (2002) Identification of Delta Helicase as the Bovine Homologue of HUPF1: Demonstration of an Interaction with the Third Subunit of DNA Polymerase Delta. Nucleic Acids Res. 30, 2232-2243.
Lu, X., Tan, C.-K., Zhou, J.-Q., You, M., Carastro, L.M., Downey, K.M., and So, A.G. (2002) Direct Interaction of Proliferating Cell Nuclear Antigen with the Small Subunit of DNA Polymerase Delta. J Biol Chem. 277, 24340-24345.
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. (2002) Delta helicase corresponds to KIAA0221: A link between DNA polymerase delta and RNA surveillance complex. Scientific World Journal 1 (Suppl 3), 77.
Strudwick, S., Carastro, L.M., Stagg, T., and Lazarus, P. (2003) Differential transcription-coupled translational inhibition of human p53 expression: A potentially important mechanism of regulating p53 expression in normal versus tumor tissue. Mol Cancer Res. 1, 463-474.
Joshi, B., Rastogi, S., Morris, M., Carastro, L. M., DeCook C., Seto, E. and Chellappan, S.P. (2007) Differential regulation of human YY1 and caspase 7 promoters by prohibitin through E2F1 and p53 binding sites. Biochem J. 401, 155-166.
Carastro, L.M., Gandikal, N., Brucklacher, R. and Lazarus P. (2007) An upstream open reading frame regulates p53 mRNA stability and translation. (Manuscript in preparation
Zhou, J.-Q., Tan, C.-K., Carastro, L.M., Downey, K.M., and So, A.G. The Small Subunit of DNA Polymerase Delta is a Counterpart of the Tau Subunit of the Escherichia coli DNA Polymerase III. Cold Spring Harbor Symposium on DNA Replication, Cold Spring Harbor, NY (September 1997).
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. Delta Helicase, a DNA Helicase that Co-Purifies with DNA Polymerase Delta. 2000 Cancer Research Poster Session, University of Miami School of Medicine-Sylvester Comprehensive Cancer Center, Miami, FL (May 2000).
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. Delta Helicase Corresponds to KIAA0221 (HUPF1): A Link Between DNA Polymerase Delta and RNA Surveillance Complex. 2001 Miami Nature Biotechnology Winter Symposium "Cell Death and Aging", Miami Beach, FL (February 2001).
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. Delta Helicase Corresponds to KIAA0221 (HUPF1): A Link Between DNA Polymerase Delta and RNA Surveillance Complex. 2001 Eastern-Atlantic Student Research Forum of the American Medical Association, Miami, FL (February 2001).
Carastro, L.M., Tan, C.-K., So, A.G., and Downey, K.M. Delta Helicase Corresponds to KIAA0221 (HUPF1): A Link Between DNA Polymerase Delta and RNA Surveillance Complex. Experimental Biology 2001, Orlando, FL (March 2001).
Carastro, L.M. and Lazarus, P. Role of Putative Upstream Reading Frames in the Translational Regulation of p53 mRNA. 95th American Association for Cancer Research National Meeting, Orlando, FL (March 2004).
Carastro, L.M. and Lazarus, P. Translation of an Upstream Open Reading Frame in p53 mRNA Triggers mRNA Decay. 96th American Association for Cancer Research National Meeting, Anaheim, CA (April 2005).
Sun, D., Duncan, K., Chen, G., Dellinger, R, Carastro, L.M., and Lazarus, P. Glucuronidation of tamoxifen and 4-hydroxytamoxifen by human UGT1A4 variants. 96th American Association for Cancer Research National Meeting, Anaheim, CA (April 2005).
Sun, D., Duncan, K., Chen, G., Dellinger, R, Carastro, L.M., Weinberg, R.B., Zhong, Q., and Lazarus, P. Glucuronidation of the active metabolites of tamoxifen by human liver microsome and individual UDP-glucuronosyltransferases (UGTs). 97th American Association for Cancer Research National Meeting, Washington, D.C. (April 2006).
Carastro, L.M., Gandikal, N., and Brucklacher, R. An uORF in p53 mRNA regulates translation and uORF-containing p53 mRNA is selectively degraded by a translation- & PIKK-dependent mechanism. Salk/Cal Tech DNA Replication & Genomic Stability 2006, Salk Institute for Biological Studies, La Jolla, CA (August 2006)
Carastro, L.M., Gandikal, N., and Brucklacher, R. An uORF in p53 mRNA regulates translation and uORF-containing p53 mRNA is selectively degraded by a translation- & PIKK-dependent mechanism. Experimental Biology 2007, Washington, D.C. (April 2007)