Bryan S. Poe and Dr. Kim L. O’Neill, Microbiology
During the course of a lifetime the human genome encounters endless assaults on its fragile genetic material, many of which are potentially teratogenic, mutagenic or even oncogenic events. To counter these genetic insults, the cell (the fundamental building unit of the human body), has in place a myriad of protective and reparative mechanisms. One of the major defenses against an existing chromosomal lesion is the nucleotide excision repair pathway.
The nucleotide excision repair (NER) pathway acts much like a transplant surgeon who removes a diseased or damaged organ and replaces it with a new functional one. Once a DNA strand is “damaged”, for example by cross linking to another strand, strand breakage, or addition of bulky alkyl or protein adducts, the site must first be recognized. In the next step the enzyme makes cuts on either side of the lesion, allowing for subsequent degradation by exonucleolytic proteins. Lastly, the excised region of nucleotides is replaced by a DNA polymerase.
For this investigation the effects of caffeine (CAF) on the NER kinetics of several different human cell lines were assayed using single cell gel electrophoresis. Exponential cultures of BELA (human cervical cancer), Raji (B cell lymphoma), and HL60 (promyelocytic leukemia) were exposed to various concentrations of CAF or received no treatment. After exposure samples from each culture were quickly treated with a isotonic solution of methyl methane sulfonate (MMS) for one hour. Each sample was subsequently washed 3 time in phosphate buffered saline (PBS) and embedded in LM agarose on individual fully frosted microscope slides. The cell membranes were then lysed in an alkaline lysis buffer containing 1.0 M NaCl and 1% lauroyl sarcosine. Immediately following lysis the salt was washed free from the samples and the slides were subjected to horizontal gel electrophoresis. After electrophoresis the samples were analyzed for DNA strand breakage using fluorescence microscopy and an integrated CCD camera/digital image analysis system.
MMS on its own induced a large number of strand breaks in all the target cell lines (Fig. 1) whereas untreated controls showed virtually no DNA fragmentation (Fig. 2). The cells that received a caffeine treatment displayed a decrease in strand breakage in response to the damaging agent (MMS) indicating that Caffeine may be playing a role in the modulation of the NER pathway (Fig. 3).
References
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