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Richard J. Calvert - One of the best experts on this subject based on the ideXlab platform.
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K Ras 4a and 4b mrna levels correlate with superoxide in lung adenocarcinoma cells while at the Protein level only mutant K Ras 4a Protein correlates with superoxide
Lung Cancer, 2013Co-Authors: Richard J. Calvert, Meghana Gupta, Anna E Maciag, Yihhorng Shiao, Lucy M. AndersonAbstract:The K-Ras gene is frequently mutated in lung and other cancers. K-Ras Protein includes two splice variants, K-Ras 4A and 4B. While K-Ras 4B is more widely expressed, recent evidence implicates K-Ras 4A in lung tumorigenesis. We found that K-Ras 4A Protein has a wide range of expression in a large panel of human lung adenocarcinoma cell lines. In cell lines with mutant K-Ras, but not those with wildtype K-Ras, the K-Ras 4A Protein had a strong positive correlation with levels of cellular superoxide. We investigated whether K-Ras 4A Protein was involved in superoxide production, or alternatively was modulated by elevated superoxide. Experiments with small interfering RNA targeting K-Ras 4A did not confirm its role in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to reduce levels of K-Ras 4A Protein. K-Ras 4A and 4B mRNA were also quantified in a number of NSCLC cell lines. 4A mRNA correlated with 4A Protein only in K-Ras-mutant cells. K-Ras 4A mRNA also correlated with superoxide, but with no difference between cell lines with mutant or wildtype K-Ras. K-Ras 4B mRNA correlated with 4A mRNA and with superoxide, in both K-Ras mutant and wildtype cells. The results are consistent with superoxide directly or indirectly up-regulating expression of all K-Ras genes, and also increasing the stability of K-Ras 4A mutant Protein selectively.
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elevated K Ras activity with cholestyramine and lovastatin but not Konjac mannan or niacin in lung importance of mouse strain
Biochemical Pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Elevated K-Ras activity with cholestyramine and lovastatin, but not Konjac mannan or niacin in lung—–Importance of mouse strain
Biochemical pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Optimization of a nonradioactive method for consistent and sensitive determination of activated K-Ras Protein.
Analytical biochemistry, 2005Co-Authors: Richard J. Calvert, Wafa Kammouni, Keith D. KikawaAbstract:Abstract Accurate measurement of activity of wild-type K-Ras Protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-worKers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transfeRase/raf-1 Ras binding domain (GST–RBD) fusion Protein attached to glutathione beads to isolate activated Ras Protein. We systematically optimized the method before use on lung tissues. Changing the GST–RBD Protein induction temperature from the original 37 to 30 °C produced a consistently greater yield of fusion Protein. To improve stability of the GST–RBD beads so as to perform large-scale experiments, 0.1% NaN3 was added. NaN3-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST–RBD beads with two commercial assay Kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST–RBD affinity method to recover activated K-Ras greatly increased the yield of fusion Protein, prolonged the useful life of GST–RBD beads to at least 24 days, and enhanced detection sensitivity.
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Lovastatin increases activated K-Ras levels, while Konjac mannan effectively reduces serum cholesterol without altering levels of activated K-Ras in lung of C56BL/6 mice.
Cancer Research, 2004Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Proc Amer Assoc Cancer Res, Volume 45, 2004 2021 A published meta-analysis of the human lipid reductions trials of the 1970’s to early ’80’s has found that the treatment subjects had a significant increase in cancer mortality (KritchevsKy & KritchevsKy, Ann Rev Nutr 12:391-416, 1992). Previous studies from our laboratory established that hypocholesterolemic agents could alter K-Ras Protein quantity and membrane versus cytoplasm distribution. K-Ras activity state was examined in the present study to define further the importance of this finding in lung carcinogenesis. K-Ras activity state in lung tissue from A/J mice (carcinogen sensitive) was examined in a previously reported abstract (AACR 2003). In the current study, lungs from a carcinogen-resistant strain (C57BL/6) were assayed for K-Ras activity state. For three weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with 25 mg/Kg of lovastatin three or five times weeKly (Lov-3X and Lov-5X). At necropsy, serum and lungs were recovered from the mice and frozen in liquid nitrogen. Serum cholesterol was determined by a commercial Kit. Activated K-Ras Protein was isolated by immunoprecipitation with a raf-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads. Total K-Ras was recovered by immunoprecipitation with a Ras antibody bound to agarose beads. After isolation, total and activated K-Ras were quantified by polyacrylamide gel electrophoresis and western blotting. Detection was performed with a K-Ras specific antibody followed by chemiluminescent visualization. Bands were quantified by densitometry. Only KM significantly reduced total serum cholesterol compared to control, Lov-5X or pair fed. K-Ras results are summarized in the table below. Only Lov-3X increased activated K-Ras levels significantly higher than control (indicated by the * signifying p = 0.0149, t-test). These results differ marKedly from our previous activated K-Ras results in male A/J mice. The presently observed lacK of increase in activated K-Ras with CS feeding contRasts with the dose-dependent increase reported earlier with the A/J mice. The present study showed a large increase in activated K-Ras with Lov feeding, in contRast to only a very small effect in the previously studied A/J mice. This may correlate with differences in tumorigenesis between mouse strains. The highly sensitive A/Js would seem perhaps more sensitive to the Known tumor promoter CS, while the relatively resistant C57BL/6 mice showed little response to CS.
David Kritchevsky - One of the best experts on this subject based on the ideXlab platform.
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elevated K Ras activity with cholestyramine and lovastatin but not Konjac mannan or niacin in lung importance of mouse strain
Biochemical Pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Elevated K-Ras activity with cholestyramine and lovastatin, but not Konjac mannan or niacin in lung—–Importance of mouse strain
Biochemical pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Lovastatin increases activated K-Ras levels, while Konjac mannan effectively reduces serum cholesterol without altering levels of activated K-Ras in lung of C56BL/6 mice.
Cancer Research, 2004Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Proc Amer Assoc Cancer Res, Volume 45, 2004 2021 A published meta-analysis of the human lipid reductions trials of the 1970’s to early ’80’s has found that the treatment subjects had a significant increase in cancer mortality (KritchevsKy & KritchevsKy, Ann Rev Nutr 12:391-416, 1992). Previous studies from our laboratory established that hypocholesterolemic agents could alter K-Ras Protein quantity and membrane versus cytoplasm distribution. K-Ras activity state was examined in the present study to define further the importance of this finding in lung carcinogenesis. K-Ras activity state in lung tissue from A/J mice (carcinogen sensitive) was examined in a previously reported abstract (AACR 2003). In the current study, lungs from a carcinogen-resistant strain (C57BL/6) were assayed for K-Ras activity state. For three weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with 25 mg/Kg of lovastatin three or five times weeKly (Lov-3X and Lov-5X). At necropsy, serum and lungs were recovered from the mice and frozen in liquid nitrogen. Serum cholesterol was determined by a commercial Kit. Activated K-Ras Protein was isolated by immunoprecipitation with a raf-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads. Total K-Ras was recovered by immunoprecipitation with a Ras antibody bound to agarose beads. After isolation, total and activated K-Ras were quantified by polyacrylamide gel electrophoresis and western blotting. Detection was performed with a K-Ras specific antibody followed by chemiluminescent visualization. Bands were quantified by densitometry. Only KM significantly reduced total serum cholesterol compared to control, Lov-5X or pair fed. K-Ras results are summarized in the table below. Only Lov-3X increased activated K-Ras levels significantly higher than control (indicated by the * signifying p = 0.0149, t-test). These results differ marKedly from our previous activated K-Ras results in male A/J mice. The presently observed lacK of increase in activated K-Ras with CS feeding contRasts with the dose-dependent increase reported earlier with the A/J mice. The present study showed a large increase in activated K-Ras with Lov feeding, in contRast to only a very small effect in the previously studied A/J mice. This may correlate with differences in tumorigenesis between mouse strains. The highly sensitive A/Js would seem perhaps more sensitive to the Known tumor promoter CS, while the relatively resistant C57BL/6 mice showed little response to CS.
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Treatment with lovastatin, cholestyramine or niacin alters K-Ras membrane association in mouse lung in a strain-dependent manner: results in females
Biochemical pharmacology, 2003Co-Authors: Richard J. Calvert, Shirley A. Tepper, Lucy M. Anderson, Bhalchandra A. Diwan, David KritchevskyAbstract:Abstract Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-Ras membrane localization, with possible pro- or anti-carcinogenic effects (K- Ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3×) or five (Lov-5×) times per weeK. After three weeKs, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-Ras Proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-Ras specific antibody. CS feeding increased membrane K-Ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-Ras, and resulted in an increase in total K-Ras in A/J and C57BL/6 mice, while again lacKing effect in Swiss mice. Niacin had no effect on K-Ras Protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-Ras Protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-Ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less liKely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.
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Alterations in membrane-bound and cytoplasmic K-Ras Protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent
Biochemical pharmacology, 2002Co-Authors: Richard J. Calvert, Shirley A. Tepper, Lucy M. Anderson, Gayatri Ramakrishna, Bhalchandra A. Diwan, David KritchevskyAbstract:Abstract Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) Ras Protein membrane localization by altering Protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25 mg/Kg of Lov three times per weeK (Lov-3X) or five times per weeK (Lov-5X). After 3 weeKs, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-Ras Proteins in lung were determined by immunoprecipitation followed by western blotting with a K-Ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-Ras and a 40% decrease in membrane K-Ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-Ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3–28%) of membrane K-Ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58–78% increase in cytoplasmic K-Ras without any reduction in the levels of membrane K-Ras. Niacin, predicted to have no effect on Ras membrane localization, decreased cytoplasmic K-Ras in A/J mice, increased both membrane and cytoplasmic K-Ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-Ras membrane localization, and potentially the risK of Ras-dependent cancer.
Lucy M. Anderson - One of the best experts on this subject based on the ideXlab platform.
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K Ras 4a and 4b mrna levels correlate with superoxide in lung adenocarcinoma cells while at the Protein level only mutant K Ras 4a Protein correlates with superoxide
Lung Cancer, 2013Co-Authors: Richard J. Calvert, Meghana Gupta, Anna E Maciag, Yihhorng Shiao, Lucy M. AndersonAbstract:The K-Ras gene is frequently mutated in lung and other cancers. K-Ras Protein includes two splice variants, K-Ras 4A and 4B. While K-Ras 4B is more widely expressed, recent evidence implicates K-Ras 4A in lung tumorigenesis. We found that K-Ras 4A Protein has a wide range of expression in a large panel of human lung adenocarcinoma cell lines. In cell lines with mutant K-Ras, but not those with wildtype K-Ras, the K-Ras 4A Protein had a strong positive correlation with levels of cellular superoxide. We investigated whether K-Ras 4A Protein was involved in superoxide production, or alternatively was modulated by elevated superoxide. Experiments with small interfering RNA targeting K-Ras 4A did not confirm its role in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to reduce levels of K-Ras 4A Protein. K-Ras 4A and 4B mRNA were also quantified in a number of NSCLC cell lines. 4A mRNA correlated with 4A Protein only in K-Ras-mutant cells. K-Ras 4A mRNA also correlated with superoxide, but with no difference between cell lines with mutant or wildtype K-Ras. K-Ras 4B mRNA correlated with 4A mRNA and with superoxide, in both K-Ras mutant and wildtype cells. The results are consistent with superoxide directly or indirectly up-regulating expression of all K-Ras genes, and also increasing the stability of K-Ras 4A mutant Protein selectively.
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elevated K Ras activity with cholestyramine and lovastatin but not Konjac mannan or niacin in lung importance of mouse strain
Biochemical Pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Elevated K-Ras activity with cholestyramine and lovastatin, but not Konjac mannan or niacin in lung—–Importance of mouse strain
Biochemical pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Lovastatin increases activated K-Ras levels, while Konjac mannan effectively reduces serum cholesterol without altering levels of activated K-Ras in lung of C56BL/6 mice.
Cancer Research, 2004Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Proc Amer Assoc Cancer Res, Volume 45, 2004 2021 A published meta-analysis of the human lipid reductions trials of the 1970’s to early ’80’s has found that the treatment subjects had a significant increase in cancer mortality (KritchevsKy & KritchevsKy, Ann Rev Nutr 12:391-416, 1992). Previous studies from our laboratory established that hypocholesterolemic agents could alter K-Ras Protein quantity and membrane versus cytoplasm distribution. K-Ras activity state was examined in the present study to define further the importance of this finding in lung carcinogenesis. K-Ras activity state in lung tissue from A/J mice (carcinogen sensitive) was examined in a previously reported abstract (AACR 2003). In the current study, lungs from a carcinogen-resistant strain (C57BL/6) were assayed for K-Ras activity state. For three weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with 25 mg/Kg of lovastatin three or five times weeKly (Lov-3X and Lov-5X). At necropsy, serum and lungs were recovered from the mice and frozen in liquid nitrogen. Serum cholesterol was determined by a commercial Kit. Activated K-Ras Protein was isolated by immunoprecipitation with a raf-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads. Total K-Ras was recovered by immunoprecipitation with a Ras antibody bound to agarose beads. After isolation, total and activated K-Ras were quantified by polyacrylamide gel electrophoresis and western blotting. Detection was performed with a K-Ras specific antibody followed by chemiluminescent visualization. Bands were quantified by densitometry. Only KM significantly reduced total serum cholesterol compared to control, Lov-5X or pair fed. K-Ras results are summarized in the table below. Only Lov-3X increased activated K-Ras levels significantly higher than control (indicated by the * signifying p = 0.0149, t-test). These results differ marKedly from our previous activated K-Ras results in male A/J mice. The presently observed lacK of increase in activated K-Ras with CS feeding contRasts with the dose-dependent increase reported earlier with the A/J mice. The present study showed a large increase in activated K-Ras with Lov feeding, in contRast to only a very small effect in the previously studied A/J mice. This may correlate with differences in tumorigenesis between mouse strains. The highly sensitive A/Js would seem perhaps more sensitive to the Known tumor promoter CS, while the relatively resistant C57BL/6 mice showed little response to CS.
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Treatment with lovastatin, cholestyramine or niacin alters K-Ras membrane association in mouse lung in a strain-dependent manner: results in females
Biochemical pharmacology, 2003Co-Authors: Richard J. Calvert, Shirley A. Tepper, Lucy M. Anderson, Bhalchandra A. Diwan, David KritchevskyAbstract:Abstract Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-Ras membrane localization, with possible pro- or anti-carcinogenic effects (K- Ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3×) or five (Lov-5×) times per weeK. After three weeKs, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-Ras Proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-Ras specific antibody. CS feeding increased membrane K-Ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-Ras, and resulted in an increase in total K-Ras in A/J and C57BL/6 mice, while again lacKing effect in Swiss mice. Niacin had no effect on K-Ras Protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-Ras Protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-Ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less liKely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.
Mary Jo Murnane - One of the best experts on this subject based on the ideXlab platform.
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N‐Ras Protein: Frequent quantitative and qualitative changes occur in human colorectal carcinomas
International Journal of Cancer, 1997Co-Authors: Sania Shuja, Mary Jo MurnaneAbstract:Point mutation and overexpression are recognized mechanisms for Ras activation in malignancy. However, little information is available on overexpression of N-Ras Protein compared with H- or K-Ras Proteins, as N-Ras-specific antibodies have only recently become available. For comparative analyses of Ras Protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K- or N-Ras Proteins. On multi-tissue blots, N-Ras Protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-Ras in colorectal function. N-Ras Protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-Ras Protein or H-Ras Protein levels. In 69% of colon carcinomas, N-Ras Protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-Ras Protein was also observed in colon cancers but less frequently (13% of cases) than N-Ras Protein. H-Ras Protein levels were too low for comparative studies. Alterations in N-Ras Protein mobility, possibly reflecting increased post-translational processing, were also detected in 42% of colon carcinomas. N-Ras Protein, typically present as a single 23 KDa band in normal mucosa, was expressed in some cancers as a 22 KDa band or as multiple bands of 20‐23 KDa. Sequencing of N-Ras DNA from 6 carcinomas with these variations in Protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-Ras Protein expression, which do not appear to correlate with the presence of typical N-Ras point mutations, result in abnormal N-Ras Protein patterns in human colorectal carcinomas. Int. J. Cancer71:767‐775, 1997. r 1997 Wiley-Liss, Inc.
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N‐Ras Protein: Frequent quantitative and qualitative changes occur in human colorectal carcinomas
International journal of cancer, 1997Co-Authors: Kwonseop Kim, Sania Shuja, Tom Kuo, Jinguo Cai, Mary Jo MurnaneAbstract:Point mutation and overexpression are recognized mechanisms for Ras activation in malignancy. However, little information is available on overexpression of N-Ras Protein compared with H- or K-Ras Proteins, as N-Ras-specific antibodies have only recently become available. For comparative analyses of Ras Protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K- or N-Ras Proteins. On multi-tissue blots, N-Ras Protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-Ras in colorectal function. N-Ras Protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-Ras Protein or H-Ras Protein levels. In 69% of colon carcinomas, N-Ras Protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-Ras Protein was also observed in colon cancers but less frequently (13% of cases) than N-Ras Protein. H-Ras Protein levels were too low for comparative studies. Alterations in N-Ras Protein mobility, possibly reflecting increased post-translational processing, were also detected in 42% of colon carcinomas. N-Ras Protein, typically present as a single 23 KDa band in normal mucosa, was expressed in some cancers as a 22 KDa band or as multiple bands of 20-23 KDa. Sequencing of N-Ras DNA from 6 carcinomas with these variations in Protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-Ras Protein expression, which do not appear to correlate with the presence of typical N-Ras point mutations, result in abnormal N-Ras Protein patterns in human colorectal carcinomas.
Shirley A. Tepper - One of the best experts on this subject based on the ideXlab platform.
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elevated K Ras activity with cholestyramine and lovastatin but not Konjac mannan or niacin in lung importance of mouse strain
Biochemical Pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Elevated K-Ras activity with cholestyramine and lovastatin, but not Konjac mannan or niacin in lung—–Importance of mouse strain
Biochemical pharmacology, 2006Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Our previous worK established that hypocholesterolemic agents altered K-Ras intracellular localization in lung. Here, we examined K-Ras activity to define further its potential importance in lung carcinogenesis. K-Ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with lovastatin 25mg/Kg three or five times weeKly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeKs, serum cholesterol was assayed with a commercial Kit. Activated K-Ras Protein from lung was affinity precipitated with a Raf-1 Ras binding domain-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads, followed by Western blotting, K-Ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-Ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-Ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-Ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-Ras activity. These data are consistent with the concept that K-Ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.
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Lovastatin increases activated K-Ras levels, while Konjac mannan effectively reduces serum cholesterol without altering levels of activated K-Ras in lung of C56BL/6 mice.
Cancer Research, 2004Co-Authors: Richard J. Calvert, Shirley A. Tepper, Wafa Kammouni, Lucy M. Anderson, David KritchevskyAbstract:Proc Amer Assoc Cancer Res, Volume 45, 2004 2021 A published meta-analysis of the human lipid reductions trials of the 1970’s to early ’80’s has found that the treatment subjects had a significant increase in cancer mortality (KritchevsKy & KritchevsKy, Ann Rev Nutr 12:391-416, 1992). Previous studies from our laboratory established that hypocholesterolemic agents could alter K-Ras Protein quantity and membrane versus cytoplasm distribution. K-Ras activity state was examined in the present study to define further the importance of this finding in lung carcinogenesis. K-Ras activity state in lung tissue from A/J mice (carcinogen sensitive) was examined in a previously reported abstract (AACR 2003). In the current study, lungs from a carcinogen-resistant strain (C57BL/6) were assayed for K-Ras activity state. For three weeKs, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% Konjac mannan (KM), or were injected with 25 mg/Kg of lovastatin three or five times weeKly (Lov-3X and Lov-5X). At necropsy, serum and lungs were recovered from the mice and frozen in liquid nitrogen. Serum cholesterol was determined by a commercial Kit. Activated K-Ras Protein was isolated by immunoprecipitation with a raf-glutathione-S-transfeRase fusion Protein bound to glutathione-agarose beads. Total K-Ras was recovered by immunoprecipitation with a Ras antibody bound to agarose beads. After isolation, total and activated K-Ras were quantified by polyacrylamide gel electrophoresis and western blotting. Detection was performed with a K-Ras specific antibody followed by chemiluminescent visualization. Bands were quantified by densitometry. Only KM significantly reduced total serum cholesterol compared to control, Lov-5X or pair fed. K-Ras results are summarized in the table below. Only Lov-3X increased activated K-Ras levels significantly higher than control (indicated by the * signifying p = 0.0149, t-test). These results differ marKedly from our previous activated K-Ras results in male A/J mice. The presently observed lacK of increase in activated K-Ras with CS feeding contRasts with the dose-dependent increase reported earlier with the A/J mice. The present study showed a large increase in activated K-Ras with Lov feeding, in contRast to only a very small effect in the previously studied A/J mice. This may correlate with differences in tumorigenesis between mouse strains. The highly sensitive A/Js would seem perhaps more sensitive to the Known tumor promoter CS, while the relatively resistant C57BL/6 mice showed little response to CS.
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Treatment with lovastatin, cholestyramine or niacin alters K-Ras membrane association in mouse lung in a strain-dependent manner: results in females
Biochemical pharmacology, 2003Co-Authors: Richard J. Calvert, Shirley A. Tepper, Lucy M. Anderson, Bhalchandra A. Diwan, David KritchevskyAbstract:Abstract Hypocholesterolemic drugs may themselves increase (cholestyramine, CS) or decrease (lovastatin, Lov) peripheral tissue de novo cholesterol biosynthesis. This will alter the abundance of prenyl groups and potentially increase (CS) or decrease (Lov) K-Ras membrane localization, with possible pro- or anti-carcinogenic effects (K- Ras is a proto-oncogene frequently mutated in lung cancer). Female A/J, Swiss, and C57BL/6 mice were fed 2 or 4% CS, 1% niacin, or injected with Lov three (Lov-3×) or five (Lov-5×) times per weeK. After three weeKs, serum cholesterol and triglycerides were determined enzymatically. Total, membrane, and cytoplasmic K-Ras Proteins were determined in lung homogenates by immunoprecipitation followed by Western blotting with a K-Ras specific antibody. CS feeding increased membrane K-Ras as hypothesized in A/J and C57BL/6 mice, but had no effect in Swiss mice. Lov failed in all three strains to reduce membrane K-Ras, and resulted in an increase in total K-Ras in A/J and C57BL/6 mice, while again lacKing effect in Swiss mice. Niacin had no effect on K-Ras Protein in any mouse strain. These results differ from our published results for male mice of the same strains, particularly for A/J mice. Increased amounts of K-Ras Protein in the membrane fraction of A/J females (but not males) treated with either Lov or CS imply that if K-Ras were to become mutated, CS could result in increased lung tumorigenesis and Lov would be less liKely to be protective in females. In the light of these data, both sexes should be included in future animal and human chemoprevention trials.
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Alterations in membrane-bound and cytoplasmic K-Ras Protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent
Biochemical pharmacology, 2002Co-Authors: Richard J. Calvert, Shirley A. Tepper, Lucy M. Anderson, Gayatri Ramakrishna, Bhalchandra A. Diwan, David KritchevskyAbstract:Abstract Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) Ras Protein membrane localization by altering Protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25 mg/Kg of Lov three times per weeK (Lov-3X) or five times per weeK (Lov-5X). After 3 weeKs, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-Ras Proteins in lung were determined by immunoprecipitation followed by western blotting with a K-Ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-Ras and a 40% decrease in membrane K-Ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-Ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3–28%) of membrane K-Ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58–78% increase in cytoplasmic K-Ras without any reduction in the levels of membrane K-Ras. Niacin, predicted to have no effect on Ras membrane localization, decreased cytoplasmic K-Ras in A/J mice, increased both membrane and cytoplasmic K-Ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-Ras membrane localization, and potentially the risK of Ras-dependent cancer.
