High-resolution magic angle spinning 1H NMR spectroscopy and reverse transcription-PCR analysis of apoptosis in a rat glioma.
The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS 1H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.
Griffin
Julian L
JL
Department of Biochemistry, University of Cambridge, UK. jlg40@mole.bio.cam.ac.uk
Blenkiron
Cherie
C
Valonen
Piia K
PK
Caldas
Carlos
C
Kauppinen
Risto A
RA
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D005910:Glioma / Q000235:genetics / Q000473:pathology*; D050356:Lipid Metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009374:Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D011522:Protons; D012334:RNA, Neoplasm / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D019014:fas Receptor
D051017:Apoptosis Regulatory Proteins; D011522:Protons; D012334:RNA, Neoplasm; D019014:fas Receptor