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Feed a fever and starve a tumor?
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TRENDS in Biochemical Sciences Vol.27 No.9 September 2002
445
Journal Club
Efficient interference Although double-stranded RNA (dsRNA)-induced gene silencing, or RNA interference (RNAi), has proven to be a useful tool for genetic manipulation in a wide range of organisms, its use in mammalian cells had been very limited until recently. More than a year ago, Tuschl and colleagues reported the effective use of short interfering RNAs (siRNAs) in mammalian cells, possibly by bypassing the antiviral response of cells usually triggered by dsRNA. These siRNAs are currently synthesized chemically, causing the cost of research to rocket. Paddison and colleagues [1] recently demonstrated their home-made short hairpin RNAs (shRNA) modeled on microRNA, to be just as effective as their synthetic counterparts in a wide range of cell lines. They generated shRNA plasmids by
cloning shRNAs directly downstream of the U6 promoter, they then transfected the cells with this plasmid, allowing functional shRNA to be transcribed in vivo. A similar approach was taken by Paul et al. [2]. RNA inserts were used once again to generate U6 snRNA promoter-driven expression cassettes, which were used in transient transfections. Miyagishi and Taira [3] extended this expression cassette system further. In their cassette, sense and antisense strands, followed by four-nucleotide overhangs, were transcribed separately by two U6 promoters. They have exploited the Epstein–Barr virus nuclear antigen 1/oriP system to maintain siRNA expression plasmids stably and extrachromosomally in cells. This study opens up a whole new world to researchers working with
mammalian cells. The exciting next step of this system would be an inducible siRNA expression cassette. The authors already indicate a potential tetracycline-inducible siRNA vector, and we look forward to this development. 1 Paddison, P.J. et al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 2 Paul, C.P. et al. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 19, 505–508 3 Miyagishi, M. and Taira, K. (2002) U6 promoterdriven siRNAs with four uridine 3’ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 19, 497–500
Isil Aksan [email protected]
Feed a fever and starve a tumor? Genotoxic damage elicits a number of cellular responses that are essential for preserving the integrity of the genome, including apoptotic cell death, cell-cycle arrest and DNA repair. Aberrant responses appear to play a role in the development of cancers, as well as the acquisition of resistance to genotoxic treatments such as radio- and chemotherapies. The mechanisms by which DNA damage can elicit programmed cell death are not well understood, but it is clear that malfunction and adaptation of these intracellular signaling pathways are associated with tumorigenesis and therapeutic resistance, respectively. Zhou et al. have used subtractive suppressive hybridization to screen changes in lymphocyte (FL5.Bcl-xL) gene expression in response to exposure to cisplatin, a genotoxic chemotherapeutic [1]. They noted significant downregulation of the gene expression for cellular bioenergetic pathways, in particular, the glucose transporters (GLUT1 and GLUT3), hexokinases (HK1 and HK2), and 6-phosphofructo-1-kinase (PFK-1). The screening results were verified by northern blots and, in the case of GLUT 1 and 3, western blots as well. The authors then compared the effects of etoposide, another http://tibs.trends.com
DNA-damaging agent, and vincristine, a non-genotoxic chemotherapeutic, on the expression of these metabolic enzymes. The results indicate that reduced expression was related to genotoxicity, as vincristine was much less effective at suppressing expression of these genes. Even so, all three compounds coordinately reduced the rates of glycolysis and oxygen consumption, suggesting a functional linkage between cytosolic and mitochondrial metabolism that can stimulate redistribution of cytochrome c, precipitating cell-cycle arrest and subsequent apoptosis. The authors conclude that changes in glucose metabolism are a common pathway for the action of genotoxic and non-genotoxic chemotherapeutics. Apparently, vincristine downregulates the bioenergetic pathways by a mechanism that does not involve the alteration of gene expression. Because of its central role in the regulation of PFK-1 activity, the authors suggested that 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase (PFK-2/FBP-2) could be involved through control of the cellular content of fructose2,6-bisphosphate (F2,6P), as the relative rates of the enzyme’s opposing activities are determined by post-translational modification (Ser/Thr phosphorylation).
These speculations correlate well with the earlier report from Minchenko et al. [2] that transformation of tumor cells is associated with a change in the relative expression of isoforms of PFK-2/FBP-2, switching to an isozyme that can maintain higher levels of F2,6P and thus support higher rates of glycolysis. The work points to a fundamental role for energy metabolism in the control of cell cycling and eventual apoptosis. This crucial role of energy metabolism in the biochemical pathways involved in the development and progression of cancer could open up new therapeutic avenues, perhaps attempting to diminish cellular fuel metabolism by targeting metabolic regulators to elicit tumor-specific cell death. 1 Zhou, R. et al. (2002), Genotoxic exposure is associated with alterations in glucose uptake and metabolism. Cancer Res. 62, 3515–3520 2 Minchenko, A. et al. (2002), Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase-3 (PFKFB3) gene. Its possible role in the Warburg effect. J. Biol. Chem. 277,6183–6187
David A. Okar [email protected]
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
TRENDS in Biochemical Sciences Vol.27 No.9 September 2002
445
Journal Club
Efficient interference Although double-stranded RNA (dsRNA)-induced gene silencing, or RNA interference (RNAi), has proven to be a useful tool for genetic manipulation in a wide range of organisms, its use in mammalian cells had been very limited until recently. More than a year ago, Tuschl and colleagues reported the effective use of short interfering RNAs (siRNAs) in mammalian cells, possibly by bypassing the antiviral response of cells usually triggered by dsRNA. These siRNAs are currently synthesized chemically, causing the cost of research to rocket. Paddison and colleagues [1] recently demonstrated their home-made short hairpin RNAs (shRNA) modeled on microRNA, to be just as effective as their synthetic counterparts in a wide range of cell lines. They generated shRNA plasmids by
cloning shRNAs directly downstream of the U6 promoter, they then transfected the cells with this plasmid, allowing functional shRNA to be transcribed in vivo. A similar approach was taken by Paul et al. [2]. RNA inserts were used once again to generate U6 snRNA promoter-driven expression cassettes, which were used in transient transfections. Miyagishi and Taira [3] extended this expression cassette system further. In their cassette, sense and antisense strands, followed by four-nucleotide overhangs, were transcribed separately by two U6 promoters. They have exploited the Epstein–Barr virus nuclear antigen 1/oriP system to maintain siRNA expression plasmids stably and extrachromosomally in cells. This study opens up a whole new world to researchers working with
mammalian cells. The exciting next step of this system would be an inducible siRNA expression cassette. The authors already indicate a potential tetracycline-inducible siRNA vector, and we look forward to this development. 1 Paddison, P.J. et al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 2 Paul, C.P. et al. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 19, 505–508 3 Miyagishi, M. and Taira, K. (2002) U6 promoterdriven siRNAs with four uridine 3’ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 19, 497–500
Isil Aksan [email protected]
Feed a fever and starve a tumor? Genotoxic damage elicits a number of cellular responses that are essential for preserving the integrity of the genome, including apoptotic cell death, cell-cycle arrest and DNA repair. Aberrant responses appear to play a role in the development of cancers, as well as the acquisition of resistance to genotoxic treatments such as radio- and chemotherapies. The mechanisms by which DNA damage can elicit programmed cell death are not well understood, but it is clear that malfunction and adaptation of these intracellular signaling pathways are associated with tumorigenesis and therapeutic resistance, respectively. Zhou et al. have used subtractive suppressive hybridization to screen changes in lymphocyte (FL5.Bcl-xL) gene expression in response to exposure to cisplatin, a genotoxic chemotherapeutic [1]. They noted significant downregulation of the gene expression for cellular bioenergetic pathways, in particular, the glucose transporters (GLUT1 and GLUT3), hexokinases (HK1 and HK2), and 6-phosphofructo-1-kinase (PFK-1). The screening results were verified by northern blots and, in the case of GLUT 1 and 3, western blots as well. The authors then compared the effects of etoposide, another http://tibs.trends.com
DNA-damaging agent, and vincristine, a non-genotoxic chemotherapeutic, on the expression of these metabolic enzymes. The results indicate that reduced expression was related to genotoxicity, as vincristine was much less effective at suppressing expression of these genes. Even so, all three compounds coordinately reduced the rates of glycolysis and oxygen consumption, suggesting a functional linkage between cytosolic and mitochondrial metabolism that can stimulate redistribution of cytochrome c, precipitating cell-cycle arrest and subsequent apoptosis. The authors conclude that changes in glucose metabolism are a common pathway for the action of genotoxic and non-genotoxic chemotherapeutics. Apparently, vincristine downregulates the bioenergetic pathways by a mechanism that does not involve the alteration of gene expression. Because of its central role in the regulation of PFK-1 activity, the authors suggested that 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase (PFK-2/FBP-2) could be involved through control of the cellular content of fructose2,6-bisphosphate (F2,6P), as the relative rates of the enzyme’s opposing activities are determined by post-translational modification (Ser/Thr phosphorylation).
These speculations correlate well with the earlier report from Minchenko et al. [2] that transformation of tumor cells is associated with a change in the relative expression of isoforms of PFK-2/FBP-2, switching to an isozyme that can maintain higher levels of F2,6P and thus support higher rates of glycolysis. The work points to a fundamental role for energy metabolism in the control of cell cycling and eventual apoptosis. This crucial role of energy metabolism in the biochemical pathways involved in the development and progression of cancer could open up new therapeutic avenues, perhaps attempting to diminish cellular fuel metabolism by targeting metabolic regulators to elicit tumor-specific cell death. 1 Zhou, R. et al. (2002), Genotoxic exposure is associated with alterations in glucose uptake and metabolism. Cancer Res. 62, 3515–3520 2 Minchenko, A. et al. (2002), Hypoxia-inducible factor-1-mediated expression of the 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase-3 (PFKFB3) gene. Its possible role in the Warburg effect. J. Biol. Chem. 277,6183–6187
David A. Okar [email protected]
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.