Date of Award

8-2007

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Susan R. Stapleton

Second Advisor

Dr. Silvia Rossbach

Third Advisor

Dr. David Reinhold

Fourth Advisor

Dr. John Spitsbergen

Abstract

DNA microarrays can be used to measure genome-wide transcript levels. These measurements may be useful in understanding cellular changes induced by a chemical agent. In this study, Affymetrix microarray technology has been used to study the effects of chloramphenicol, an antibiotic that inhibits bacterial and mitochondrial protein synthesis, on the transcription profile in mammalian cells. Transcript levels in BB88 murine erythroleukemia cells treated with 50 micromolar ( mu M) chloramphenicol, a concentration shown to inhibit BB88 proliferation, are measured. Using total RNA from treated cells, biotin-labeled cRNA was prepared and hybridized to Affymetrix U74Av2 microarrays. From these hybridizations, expression levels were measured for approximately 12,000 transcripts. Methods to identify genes whose transcript levels increase or decrease during the treatments are described. In particular, chloramphenicol increases transcript levels for a subunit of cytochrome c oxidase (COX), a complex metalloprotein that plays a critical role in cellular respiration. Specifically, transcripts for cytochrome c oxidase VIa-H (COX VIa-H), a nuclear-encoded subunit of complex IV in the mitochondrial respiratory chain, increase greater than 20-fold after 24 hours of chloramphenicol treatment. While transcripts for COX VIa-H are increased in chloramphenicol-treated cultured erythroleukemia cells, transcripts for other nuclear encoded COX subunits appear unaffected. To follow-up on these findings, a reverse transcription-polymerase chain reaction (Tagman super( registered ) PCR) method has been used to confirm that chloramphenicol causes specific increases in COX VIa-H expression in these cells not shown previously to contain COX VIa-H. Further, these results have been extended by showing that increases in the COX VIa-H transcript by chloramphenicol treatment are concentration- and time-dependent. Finally, in cells treated with other compounds that inhibit either mitochondrial protein synthesis or respiration, COX VIa-H transcripts are increased. While chloramphenicol's mechanism of mammalian cell toxicity remains uncertain, these findings support evidence that shows chloramphenicol has an affect on mitochondrial proteins and suggests the COX VIa-H transcript may be an anti-proliferation marker (biomarker) in these cells.

Comments

5th Advisor: Dr. Rolf Kletzien

Access Setting

Dissertation-Open Access

Download

Included in

Chemistry Commons

Share

COinS