Raman microscopy of bladder cancer cells expressing green fluorescent protein.
Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.
University of Michigan, School of Dentistry, Department of Biologic and Materials Sciences, 1011 North University Avenue, Ann Arbor, Michigan 48109-1078, United States.
Gurjit S
GS
Mandair
University of Michigan, Department of Urology and Biointerfaces Institute, NCRC Building 20, 2800 Plymouth Road, Ann Arbor, Michigan 48109-2800, United States.
Amy L
AL
Han
University of Michigan, Department of Urology and Biointerfaces Institute, NCRC Building 20, 2800 Plymouth Road, Ann Arbor, Michigan 48109-2800, United States.
Evan T
ET
Keller
University of Michigan, Department of Chemistry, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, United States.
Michael D
MD
Morris
D045744:Cell Line, Tumor; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods*; D001743:Urinary Bladder / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000737:chemistry / Q000000981:diagnostic imaging* / Q000235:genetics / Q000378:metabolism*
D049452:Green Fluorescent Proteins