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Benjamin Darbro


benjamin-darbro@uiowa.edu
Mentor: Aloysius Klingelhutz, Ph.D.
Lab Phone: 353-5682

The Mechanism of Telomere-Independent Cellular Senescence in Human Keratinocytes

After a finite number of cell divisions, normal somatic cells enter a non-proliferative state referred to as senescence. Following introduction of the enzyme telomerase some cell types can bypass senescence and become immortal. Other cell types, however, including human keratinocytes, experience a telomere-independent senescence that cannot be overcome by maintaining telomere length. Human keratinocytes positive for telomerase activity are immortalized upon acquisition of a loss-of-function mutation involving the cyclin dependent kinase inhibitor p16INK4a. This finding, in addition to the observation that p16 protein levels continue to increase as keratinocytes age in culture, suggests a role for the p16/Rb pathway in keratinocyte telomere-independent senescence. It is our hypothesis that the increased p16 expression exhibited during in vitro keratinocyte aging, and consequent telomere-independent senescence, is the result of transcriptional activation of the p16 promoter induced in response to a reepithelialization signal. Preliminary evidence obtained in our laboratory with multiple primary keratinocyte cell strains has verified a delay in p16 protein accumulation and an extended lifespan when keratinocytes are grown in co-culture with post-mitotic fibroblast feeder cells. Further data obtained using DNA oligonucleotide microarrays, and subsequent validation, has shown that mRNA levels of several genes involved in keratinocyte differentiation are increased in keratinocytes co-cultured with feeder cells while mRNAs that code for proteins involved in keratinocyte reepithelialization, a component of the wound healing process, are induced in keratinocytes cultured on plastic alone. Our data indicate that culture conditions that favor keratinocyte migration and proliferation, common components of the wound healing response, lead to a rapid increase in p16 protein levels and consequent telomere-independent senescence. We are currently performing EMSAs and promoter expression studies to determine how p16 is transcriptionally upregulated during this process. This work will provide insight into the role of p16 in the development of cancer and cellular aging.

Abstracts:

Darbro, B.W., Klingelhutz, A.J. The Keratinocyte Wound Healing Response and Mechanism of p16INK4a Induced Telomere-Independent Senescence. Student Interdisciplinary Health Research Poster Session, University of Iowa. April 2004. (Poster Presentation)

Darbro, B.W., Klingelhutz, A.J. The Keratinocyte Wound Healing Response and Mechanism of p16INK4a Induced Telomere-Independent Senescence. Carver College of Medicine Research Week, University of Iowa. April 2004. (Poster Presentation)

Darbro, B.W., Klingelhutz, A.J. Telomere-Independent Senescence is Associated with Activation of a Re-epithelialization Response in Human Keratinocytes. AACR 95th Annual Meeting, Orlando, FL. March 2004. (Oral Presentation)

Darbro, B.W., Klingelhutz, A.J. The Mechanism of p16INK4a Induced Telomere-Independent Senescence in Human Keratinocytes. Molecular Biology Retreat, Strawberry Point, IA. October 2003. (Oral Presentation)

Darbro, B.W., Klingelhutz, A.J. The Mechanism of p16INK4a Induced Telomere-Independent Senescence in Human Keratinocytes. Medical Student Research Day, University of Iowa. September 2003. (Oral Presentation)

Darbro, B.W., Klingelhutz, A.J. The Mechanism of p16INK4a Induced Telomere-Independent Senescence in Human Keratinocytes. MSTP Retreat, Oakdale Campus, University of Iowa. August 2003. (Oral Presentation)

Darbro, B.W., Klingelhutz, A.J. The Mechanism of Telomere-Independent Cellular Senescence in Human Keratinocytes. Carver College of Medicine Research Week, University of Iowa. April 2003. (Poster Presentation)

Darbro, B.W., Klingelhutz, A.J. Analysis of Gene Expression Patterns Associated with Cellular Senescence and p16INK4a Expression. National MD/PhD Conference, Aspen, CO. July 2002. (Poster Presentation)

Darbro, B.W. Generation X and the Cloning Age: From Pig Oocytes to Immortalization. Cloning: Bane or Blessing, Oak Ridge, TN. October 2000. (Oral Presentation)

Publications:

Darbro BW, Lee KM, Nguyen NK, Domann FE, Klingelhutz AJ. Methylation of the p16(INK4a) promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells. Oncogene. 2006 Nov 30;25(56):7421-33. Epub 2006 Jun 12. PubMed PMID: 16767161; PubMed Central PMCID: PMC1894570.

Darbro BW, Schneider GB, Klingelhutz AJ. Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes. J Invest Dermatol. 2005 Sep;125(3):499-509. PubMed PMID: 16117791; PubMed Central PMCID: PMC2020850.

Klingelhutz AJ, Qian Q, Phillips SL, Gourronc FA, Darbro BW, Patil SR. Amplification of the chromosome 20q region is associated with expression of HPV-16 E7 in human airway and anogenital epithelial cells. Virology. 2005 Sep 30;340(2):237-44. PubMed PMID: 16051300; PubMed Central PMCID: PMC2223067.



Honors and Awards

  • College of Medicine Research Week (2003) Center on Aging Award signifying recognition for presenting
  • Teaching Assistant for Principles of Infectious Disease Lab (2002 and 2003) Gross Anatomy Tutor (20
  • American Medical Student Association Iowa Medical Society National Residence Hall Honorary (NRHH
  • College of Medicine Research Week-Iowa City, IA (2003) National MD/PhD Conference-Aspen, CO (2002)