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Annals of Clinical & Laboratory Science 36:185-193 (2006)
© 2006 Association of Clinical Scientists

Fatty Acid Synthase and its mRNA Concentrations Are Decreased at Different Times Following Hoechst 33342-induced Apoptosis in BC3H-1 Myocytes

Xinbo Zhang and Frederick L. Kiechle
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan

Address correspondence to Frederick L. Kiechle, M.D., Ph.D., 18811 Riverside Drive, Beverly Hills, MI 48025, USA; tel 248 646 2724; fax 248 551 3694; e-mail fkiechle{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 µg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 µg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 µg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis.

Keywords: Hoechst 33342, apoptosis, BC3H-1 myocytes, fatty acid synthase, RT-PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fatty acid synthase (FAS, EC 2.3.1.85 [EC] ) is the sole enzyme responsible for the de novo biosynthesis of fatty acids from the condensation of acetyl-CoA and malonyl-CoA. Mammalian FAS is a complex multifunctional enzyme that contains seven catalytic domains and a 4'-phosphopantetheine prosthetic group on a single 260,000-dalton poly-peptide [1]. Recent studies demonstrate that FAS plays an important role in intracellular processes, such as apoptosis and proliferation, as well as a role in energy homeostasis by converting excess carbon intake into fatty acids for storage [2]. In most normal human tissues, FAS is expressed at low to undetectable levels due to the presence of dietary fatty acids.

The nutritional and hormonal regulation of FAS is primarily at the level of transcription [3,4]. For example, prolactin inhibits the expression of FAS by activating STAT5A binding to the rat FAS promoter [4]. In contrast, FAS is overexpressed in a large number of human cancers despite high levels of endogenous fatty acids [5]. For example, high levels of FAS expression and activity have been reported in preneoplastic lesions of the prostate [6,7], stomach [8], esophagus [9], oral cavity [10], colon [11,12], and breast [13,14]. In addition, FAS inhibition leads to cytostatic and cytotoxic effects in cultured tumor cells, and significant antitumor effects in both human breast and prostate cancer xenografts [15–20]. Therefore, pharmacological inhibition of tumor-associated FAS hyperactivity is under investigation as a potential chemotherapeutic target in established carcinomas.

Hoechst 33342 is a compound that induces apoptosis in both non-transformed [21,22] and transformed cells [23–33]. The mechanism by which Hoechst 33342 induces apoptosis involves multiple intracellular events including mitochondrial dysfunction, disruption of 3 complexes: TATA box binding protein/TATA box, replication protein A/single-stranded DNA, and topoisomerase I/DNA cleavable complexes [34]. Hoechst 33342-induced apoptosis is associated with an increased intracellular concentration of E2F-1 transcription factor and nitric oxide concentration [34]. To pursue the mechanism of Hoechst 33342-induced apoptosis, we have evaluated intracellular changes in FAS protein and FAS mRNA concentration, and alterations of FAS-mediated lipid metabolism during Hoechst 33342-induced apoptosis in BC3H-1 myocytes. In the present study, our results indicate that FAS protein, FAS mRNA concentrations, and lipid anabolism are significantly decreased in a dose- and time-dependent fashion during Hoechst 33342-induced apoptosis in BC3H-1 myocytes, suggesting that the failure of lipid anabolism is associated with Hoechst 33342-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture.  The murine muscle cell line (BC3H-1) was grown in Dulbecco’s modified Eagles medium (DMEM) (Mediatech, Herndon, VA) with 10% fetal bovine serum (FBS) (Biocell Laboratories, Rancho Dominguez, CA) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air as previously described [35]. For experiments, cells were plated at a density of 1 x 104 cells/ml and were cultured for 2 days with 80–90% confluence prior to treatment with Hoechst 33342 (Sigma, St Louis, MO). Hoechst 33342 and Hoechst 33258 were dissolved in distilled water at 25 mg/ml and added to DMEM with 2% FBS at either a final concentration of 20 µg/ml for incubation with cells for 0, 0.5, 1, 2, 3, 6, 12, or 24 hr or different concentrations of Hoechst 33342 (0, 0.25, 0.5, 2.5, 5, 10, and 20 µg/ml).

Metabolic [1-14C]-acetate-labeling of lipids.  For fatty acid synthesis, BC3H-1 myocytes were incubated with DMEM with 2% FBS containing 0.5 µCi/ml [1-14C]-acetate in the presence of either different concentrations of Hoechst 33342 (0, 0.5, 2.5, 10, and 20 µg/ml) for 24 hr or 20 µg/ml for different times (0, 0.5, 1, 3, 6, 12, and 24 hr). The untreated and treated cells were harvested for lipid analysis [36]. For pulse-chase studies, BC3H-1 myocytes were pulsed with 0.5 µCi/ml [1-14C]-acetate in DMEM with 2% FBS for 24 hr, then washed 3x with phosphate-buffered saline (PBS), and further incubated with cold DMEM with 2% FBS in the presence of 20 µg/ml Hoechst 33342 for different times (0, 0.5, 1, 3, 6, 12, and 24 hr). The treated cells were harvested for lipid analysis [36].

Total lipid purification and lipid analysis.  The total cellular lipids of the samples were extracted with chloroform-methanol [37]. Different lipid components were analyzed by high performance thin layer chromatography (HPTLC). Phospholipid species were separated with chloroform:methanol:water (60:30:5, v/v) to 7 cm, and hexane:diethyl ether:acetic acid (80:20:1.5, v/v) to 14 cm of the plate [38]. The radioactivity of the total lipid samples was quantified by liquid scintillation counting and the HPTLC plates were subjected to autoradiography. For the [1-14C]-acetate unlabeled groups, the total lipids of untreated and treated cells were extracted with chloroform-methanol [37], total phospholipids were developed by chloroform:methanol:acetic acid:formic acid:water (70:30: 12:4:2, v/v). The HPTLC plates were visualized by spraying with 3% cupic acetate in 8% phophoric acid solution and heating at 120°C for 15 min [39].

Measurement of intracellular FAS concentration by Western blotting.  Untreated and treated cells were washed 2x with phosphate-buffered saline (PBS), scraped from flasks, and harvested at 1,000 rpm by centrifugation. The cells were suspended in 250 µl of boiling sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 2% ß-mercaptoethanol), boiled for an additional 5 min, and then passed several times through a 26-gauge needle. Samples were electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20) for 1 hr at room temperature. A 1:250 dilution of anti-mouse FAS IgG (Pierce, Rockford, IL) was added to this solution and primary hybridization was conducted at room temperature for 1 hr. After 3 successive washings in 1x TBST (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween-20), a goat anti-mouse IgG-horseradish peroxidase conjugate (1:2000 dilution) (Pierce, Rockford, IL) in 1x TBST was added for 1 hr at room temperature. After 3 washes with 1x TSBT, chemiluminescence was used to detect the horseradish peroxidase conjugate after exposure of X-ray film [31]. Each band was quantitated by densitometry.

Measurement of intracellular mRNA concentration by real-time RT-PCR.  Quantitative real-time PCR was carried out to detect glyceraldehyde-3-phosphate dehydrogense (GAPDH) expression, which was used to normalize the amount of cDNA of each sample. GAPDH primers were:

5'-AACTTTGGCATTGTGGAAGG-3' and

5'-GGATGCCGGGATGATGTTCT-3'.

Equal amounts of FAS cDNA from each sample were amplified using the following primers:

5'-ATTGCATCAAGCAAGTGCAG-3' and

5'-GAGCCGTCAAACAGGAAGAG-3'.

Total RNA from treated and untreated BC3H-1 myocytes was purified by use of a total RNA purification kit (Gentra, Minneapolis, MN). FAS mRNA concentrations of samples were analyzed using an ABI Prism 7000 cycler (Applied Biosystems, Foster City, CA) with Sybr-green fluorochrome (Giagen, Valencia, CA). PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hoechst 33342 on the incorporation of [1-14C]-acetate into phospholipids.  Radiolabeled acetate has long been used for the measurement of lipid and cholesterol synthesis in biochemistry [40,41]. Acetate is easily taken up by cells and converted to acetyl-CoA in both the cytosol and mitochondria by acetyl-CoA synthetase [32–44], a common metabolic intermediate for synthesis of cholesterol and fatty acids [45,46]. At the same time, acetyl-CoA is also a substrate for the TCA cycle [44]. Acetyl-CoA is one of the substrates for the de novo synthesis of long-chain fatty acid catalyzed by FAS, which is a NADPH-dependent reaction [1]. In the present study, [1-14C]-acetate was used as a radiotracer to detect the change in lipid metabolism during Hoechst 33342-induced apoptosis in BC3H-1 myocytes. Our results show that the amount of [1-14C]-acetate incorporation into total lipids and phospholipids is significantly decreased by lethal doses of Hoechst 33342 (10 and 20 µg/ml) (Figs. 1A,BGo, 2A,BGo). [1-14C]-Acetate incorporation into total lipids and phospholipids remains stable in sub-lethal dose-treated groups (0.5, 2.5, 10 µg/ml), and immediately decreases to >70% of [1-14C] incorporation in the control group or the sub-lethal dose groups at lethal doses (10, 20 µg/ml). A similar dose-response pattern has been observed in Hoechst 33342-induced decrease of topoisomerase I activity [24,29], TATA box-binding protein complex formation [25,26], replication protein A complex formation [31], and luciferase activity [47] during Hoechst 33342-induced apoptosis. These findings suggest that one of the features of Hoechst 33342-induced apoptosis is rapid dysfunction of numerous proteins or enzymes.


Figure 1
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  		Fig.1. Effect of different concentrations of Hoechst 33342 on the incorporation of [1-14C]-acetate into total lipids and phospholipids. BC3H-1 myocytes were incubated with DMEM with 2% FBS containing 0.5 µCi/ml [1-14C]-acetate in the presence of different concentrations of Hoechst 33342 (0, 0.5, 2.5, 10, and 20 µg/ml) for 24 hr. The treated cells were harvested and the total cellular lipids of the samples were extracted with chloroform-methanol. The radioactivities of the total lipid samples were quantified by a liquid scintillation counter and the phospholipid lipid components were analyzed by HPTLC and the HPTLC plates were subjected to autoradiography. A (left panel), radioactivity of the incorporation of [1-14C]-acetate into total lipids; B (right panel), HPTLC autoradiogram of phospholipids. ** p <0.001

 

Figure 2
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  		Fig. 2. Effect of different treatment time of 20 µg/ml Hoechst 33342 on the incorporation of [1-14C]-acetate into total lipids and phospholipids. BC3H-1 myocytes were incubated with DMEM with 2% FBS containing 0.5 µCi/ml [1-14C]-acetate in the presence of 20 µg/ml Hoechst 33342 for different times (0, 0.5, 1, 3, 6, 12, and 24 hr). The treated cells were harvested and the total cellular lipids of the samples were extracted with chloroform-methanol. The radioactivities of the total lipid samples were quantified by a liquid scintillation counter and the phospholipid lipid components were analyzed by HPTLC and the HPTLC plates were subjected to autoradiography. A (left panel), radioactivity of the incorporation of [1-14C]-acetate into total lipids; B (right panel), HPTLC autoradiogram of phospholipids. ** p <0.001

 
Effect of Hoechst 33342 on catabolism of [1-14C]-labeled total lipids and phospholipids.  To further determine the fate of intracellular total lipids and phospholipids during Hoechst 33342-induced apoptosis, pulse-chase experiments were employed. The results show that the rate of both reduction in [1-14C]-labeled total lipids and phospholipids are very slow in the 20 µg/ml Hoechst 33342 treatment group and the control group after 24 hr treatment, suggesting that Hoechst 33342-decreased incorporation of [1-14C]-acetate into lipids is mainly due to decrease in de novo fatty acid biosynthesis (Fig. 3A,BGo). Although 20 µg/ml Hoechst 33342 reduces the concentration of [1-14C]-acetate labeled lipids after 12 hr treatment (Fig. 3AGo), thin layer chromatography results indicate that there are no changes in the individual phospholipids when compared to the control group (Fig. 3BGo). However, using [1-14C] acetate-unlabeled BC3H-1 myocytes, Hoechst 33342 decreased the concentration of total cell phospholipids in a dose- and time-dependent manner. Significant decreases were noted for phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin (Fig. 4A,BGo). These findings suggest that Hoechst 33342-induced apoptosis is mainly associated with inhibition of anabolism of total lipid synthesis, but has no effect on catabolism of total lipid synthesis.


Figure 3
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  		Fig. 3. Effect of 20 µg/ml Hoechst 33342 on the catabolism of [1-14C]-acetate-labeled total lipids and phospholipids. BC3H-1 myocytes were incubated with DMEM with 2% FBS containing 0.5 µCi/ml [1-14C]-acetate for 24 hr, then washed 3x with phosphate-buffered saline (PBS), and further incubated with cold DMEM with 2% FBS in the presence of 20 µg/ml for different times (0, 0.5, 1, 3, 6, 12, and 24 hr). The treated cells were harvested and the total cellular lipids of the samples were extracted with chloroform-methanol. The radioactivities of the total lipid samples were quantified by a liquid scintillation counter and the phospholipid lipid components were analyzed by HPTLC and the HPTLC plates were subjected to autoradiography. A (left panel), radioactivity of the total lipids; B (right panel), HPTLC autoradiogram of phospholipids. ** p <0.001

 

Figure 4
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  		Fig. 4. Effect of Hoechst 33342 on the synthesis of unlabeled phospholipids. BC3H-1 myocytes were incubated with DMEM with 2% FBS containing either different concentrations of Hoechst 33342 (0, 2.5, 10, 20 µg/ml) for 24 hr A (left panel), or 20 µg/ml Hoechst 33342 for different times (0, 0.5, 1, 3, 6, 12, and 24 hr); B (right panel) The treated cells were harvested and the total cellular lipids were extracted with chloroform-methanol. The phospholipid lipid components in samples were analyzed by HPTLC.

 
Effect of Hoechst 33342 on intracellular FAS protein concentrations.  To study whether inhibitory de novo fatty acid synthesis caused by lethal doses of Hoechst 33342 is associated with inhibition of FAS activity and/or degradation of FAS protein, intracellular FAS concentration was measured by Western blottting. Our results indicate that the intracellular FAS protein concentration becomes undetectable after 10 or 20 µg/ml treatment for 24 hr (Fig. 5A,BGo), and that intracellular FAS protein concentration is rapidly decreased after the lethal dose (20 µg/ml) treatment with Hoechst 33342 for 1 hr, and becomes undetectable after 6 hr of treatment (Fig. 6A,BGo). Although sublethal doses significantly decrease intracellular FAS protein concentrations after 24 hr treatment, the decrease in intracellular FAS protein concentrations is not associated with Hoechst 33342-induced apoptosis.


Figure 5
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  		Fig. 5. Effect of different concentrations of Hoechst 33342 on intracellular FAS protein concentrations. BC3H-1 myocytes were incubated with DMEM with 2% FBS in the presence of different concentrations of Hoechst 33342 (0, 0.5, 1, 2.5, 10, and 20 µg/ml) for 24 hr. The treated cells were harvested and the total cellular proteins of the samples were extracted. Protein samples were electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The horseradish peroxidase conjugate antibody detected FAS protein bands after exposure of X-ray film. The density of each band was quantitated. A (left panel), comparison of the density of each FAS band; B (right panel), result of Western blot. * p <0.05; ** p <0.001

 

Figure 6
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  		Fig. 6. Effect of different treatment time of 20 µg/ml Hoechst 33342 on intracellular FAS protein concentrations. BC3H-1 myocytes were incubated with DMEM with 2% FBS in the presence of 20 µg/ml Hoechst 33342 for different times (0, 0.5, 1, 3, 6, 12, and 24 hr). The treated cells were harvested and the total cellular proteins of the samples were extracted. Protein samples were electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The horseradish peroxidase conjugate antibody detected FAS protein bands after exposure of X-ray film. The density of each band was quantitated. A (left panel), comparison of the density of each FAS band; B (right panel), result of Western blot. A represents control cells and B represents 20 µg/ml Hoechst 33342-treated cells. * p<0.05; ** p<0.001

 
These findings suggest that the decrease in the FAS intracellular concentration results in decreased incorporation of [1-14C]-acetate into total lipids and phospholipids. Therefore, Hoechst 33342-increased reduction in the incorporation of [1-14C]-acetate into lipids is attributable to the rapid decrease in intracellular FAS protein concentration. Intracellular FAS protein degradation may be mediated by a protease, such as caspase 3. Jerome and Chen [21] demonstrated that Hoechst 33342-induced apoptosis is associated with activation of caspase 3.

Effect of Hoechst 33342 on intracellular FAS mRNA concentration.  To illustrate the correlation of Hoechst 33342-decreased FAS protein and FAS mRNA concentration in the BC3H-1 myocytes, intracellular FAS mRNA concentrations are measured by real-time RT-PCR. The intracellular FAS mRNA concentration is decreased more than 3 cycles and 5 cycles after 20 µg/ml Hoechst 33342 treatment for 12 and 24 hr when compared to the control and the sublethal Hoechst 33342 treatment groups (Fig. 7AGo). Sublethal dose treatment of Hoechst 33342 for 24 hr does not alter intracellular FAS mRNA concentration when compared to the control group, but the intracellular FAS mRNA concentration in 20 µg/ml Hoechst 33342 treatment for 24 hr decreases 4 cycles. The intracellular concentration of GAPDH mRNA also decreases after 20 µg/ml Hoechst 33342 treatment for 24 hr (data not shown).


Figure 7
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  		Fig. 7. Effect of different treatment times of 20 µg/ml Hoechst 33342 on intracellular FAS mRNA concentrations. BC3H-1 myocytes were incubated with DMEM with 2% FBS in the presence of 20 µg/ml Hoechst 33342 for different times (0, 0.5, 1, 2, 3, 6, 12, and 24 hr). Total RNA of the treated cells was purified. FAS mRNA concentrations of samples were analyzed using the ABI Prism 7000 cycler. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. A (upper panel), amplification plots; B (lower panel), ethidium bromide gel.

 
Theoretically, one cycle difference between two samples indicates a 2-fold mRNA concentration difference between the two samples. Therefore, 20 µg/ml Hoechst 33342 decreases the intracellular FAS mRNA and GAPDH mRNA concentration by 8 to 10 fold after 24 hr treatment when compared to the control and the sublethal treatment groups. However, argrose gel results of real-time RT-PCR products indicate that the differences of densities among samples are not clearly seen (Figs. 7BGo,8Go,9Go), suggesting that real-time RT-PCR is more reliable for the identification of differences in intracellular mRNA concentration.


Figure 8
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  		Fig. 8. Effect of different concentrations of Hoechst 33342 on intracellular FAS mRNA concentrations. BC3H-1 myocytes were incubated with DMEM with 2% FBS in the presence of different concentrations of Hoechst 33342 (0, 0.25, 0.5, 1, 2.5, 5, and 20 µg/ml) for 24 hr. Total RNA of the treated cells was purified. FAS mRNA concentrations of samples were analyzed using the ABI Prism 7000 cycler. PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

 

Figure 9
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  		Fig. 9. Effect of different concentrations of Hoechst 33342 on intracellular GAPDH concentrations. BC3H-1 myocytes were incubated in the presence of different concentrations of Hoechst 33342 for 24 hr. Total RNA was purified. GAPDH mRNA concentrations of samples were analyzed and PCR products were separated and visualized as noted in Fig. 8Go.

 
In conclusion, the reduction in intracellular FAS mRNA concentration induced by a lethal dose Hoechst 33342 (20 µg/ml) occurs more than 6 hr after inhibition of fatty acid synthesis and degradation of FAS protein. These findings suggest that inhibition of fatty acid synthesis caused by lethal dose of Hoechst 33342 (20 µg/ml) is dependent on the degradation of intracellular FAS protein, but not on reduction of intracellular FAS mRNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis, or programmed cell death, describes a form of cell death that follows a precise genetic program. This type of cell death is evolutionarily conserved and includes activation of "death" proteases, called caspases. Caspase and nuclease activation is fundamental for the morphological and biochemical changes in the nuclei and cells observed during apoptosis [34,48]. Apoptosis is a complex event that is involved in multiple signaling transduction pathways and organelles such as mitochondria [34,48,49]. Hoechst 33342-induced apoptosis has been documented by investigations in Kiechle’s laboratory [23–33], but the precise mechanism of Hoechst 33342-induced apoptosis remains unclear. The present study explores the effect of Hoechst 33342-induced apoptosis in BC3H-1 myocytes on the synthesis of total lipids and phospholipids. There is evidence to demonstrate that lipid degradation is an early event during apoptosis and that some lipid degradation products are used as a substrate to synthesize new lipid components to trigger apoptosis. For example, De Maria et al [50] reported that the apoptotic signal triggered by CD95 cross-linking increases hydrolysis of sphingomyelin to produce ceramide, which is followed by increased synthesis of ganglioside GD3. GD3 synthesis is rapid, transient, and peaks 15 min after CD95 stimulation. Phospholipids such as phosphatidylcholine and cardiolipin also play important roles in different apoptotic pathways [51,52]. Therefore, alterations in the metabolism of total lipids and phospholipids during Hoechst 33342-induced apoptosis may be relevant to understanding its signal transduction pathway.

Our present results demonstrate that lethal doses of Hoechst 33342 significantly inhibit the synthesis of total lipid and individual phospholipids, but have no effect on the degradation of total lipids and phospholipids. Also, lethal doses of Hoechst 33342 significantly decrease the intracellular FAS protein concentration, which is followed more than 6 hr later by a decrease of intracellular FAS mRNA concentration. Therefore, the rapid degradation of intracellular FAS protein is attributable to the inhibition of fatty acid synthesis observed. Sublethal doses of Hoechst 33342 do not significantly inhibit fatty acid synthesis or modify intracellular FAS protein or FAS mRNA synthesis. Hoechst 33342 induces apoptosis but does not inhibit FAS activity. The inhibition of fatty acid synthesis can induce cancer cell apoptosis [15–20]. Therefore, activation of proteases that mediate FAS protein degradation may result in the initiation of apoptosis in cancer cells.

The mechanism whereby inhibition of fatty acid synthesis produces its antitumor effect remains unexplained. Similarly, the role of increased endogenous fatty acid synthesis in tumorigenesis is unknown, although fatty acids are implicated in tumorigenesis [53], as trophic factors [54,55], in receptor-mediated signal transduction [56], and as modulators of tumor cell adhesion [57]. However, it does appear that tumor cells have an obligatory requirement for endogenous fatty acid synthesis whereas normal cells do not, which might provide new strategies for cancer chemotherapy targeted to the fatty acid synthesis pathway.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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