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rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

Melichar B, Hu W, Patenia R, Melicharová K, Gallardo ST, Freedman R - J Transl Med (2003)

Bottom Line: However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells.CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma.Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynecologic Oncology, The University of Texas, M,D, Anderson Cancer Center, Houston, Texas, U,S,A. rfreedma@mdanderson.org

ABSTRACT
BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

No MeSH data available.


Related in: MedlinePlus

Cytostatic activity of IFN-γ, NG-hydroxy-L-arginine and the effect of polyamines on IFN-γ-induced cytostasis. NMP-1 cells were seeded at 104 cells/well into 96-well plates in RPMI 1640 with 10% fetal calf serum and cultured overnight. The next day, the cells were washed with phosphate buffered salines, and the medium was replaced with a low-arginine, serum-free medium (5% bovine serum albumin, 1.5% Iscove's modified Dulbecco's medium [GIBCO], 0.2% glucose, 0.01% magnesium sulfate, 0.016% calcium chloride, transferrin 5.5 μg/ml, and insulin 4 μg/ml in Hank's balanced salt medium [GIBCO], pH 7.4) alone or with rIFN-γ (500 U/ml). The arginine concentration of this medium was 6 μM, lower than plasma concentration (normal range, 50–126 μM [38]. After another 24 hours, rIFN-γ (500 U/ml), NG-hydroxy-L-arginine (250 μM), or rIFN-γ plus ornithine (1 mM), putrescine (1 mM), or spermidine (0.5 mM) was added. The cells were then pulsed with [3H] thymidine and incubated for another 24 hours. The cytostatic activity was determined as described in Materials and Methods. Similar results were observed with other EOC cell lines and when experiments were done with RPMI 1640 supplemented with 10% fetal calf serum. Data shown are representative of 3 separate experiments.
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Figure 3: Cytostatic activity of IFN-γ, NG-hydroxy-L-arginine and the effect of polyamines on IFN-γ-induced cytostasis. NMP-1 cells were seeded at 104 cells/well into 96-well plates in RPMI 1640 with 10% fetal calf serum and cultured overnight. The next day, the cells were washed with phosphate buffered salines, and the medium was replaced with a low-arginine, serum-free medium (5% bovine serum albumin, 1.5% Iscove's modified Dulbecco's medium [GIBCO], 0.2% glucose, 0.01% magnesium sulfate, 0.016% calcium chloride, transferrin 5.5 μg/ml, and insulin 4 μg/ml in Hank's balanced salt medium [GIBCO], pH 7.4) alone or with rIFN-γ (500 U/ml). The arginine concentration of this medium was 6 μM, lower than plasma concentration (normal range, 50–126 μM [38]. After another 24 hours, rIFN-γ (500 U/ml), NG-hydroxy-L-arginine (250 μM), or rIFN-γ plus ornithine (1 mM), putrescine (1 mM), or spermidine (0.5 mM) was added. The cells were then pulsed with [3H] thymidine and incubated for another 24 hours. The cytostatic activity was determined as described in Materials and Methods. Similar results were observed with other EOC cell lines and when experiments were done with RPMI 1640 supplemented with 10% fetal calf serum. Data shown are representative of 3 separate experiments.

Mentions: rIFN-γ produced significant growth inhibition in 7 of 8 cell lines examined, the exception being SKOV3, a cell line with negligible arginase activity (Table 1). Since the growth-inhibitory activity of rIFN-γ in certain EOC lines appeared to be accompanied with arginase inhibition, and since formation of ornithine from arginine catalyzed by arginase represents the first step in the synthesis of polyamines, we hypothesized the inhibition of arginase activity might be at least partly responsible for the growth-inhibitory effects of rIFN-γ on EOC cell lines. However, the addition of polyamines, ornithine, putrescine, or spermidine did not abolish the growth-inhibitory activity of rIFN-γ (Fig. 3). In addition, there was no interference with growth inhibition by rIFN-γ in EOC cell lines after treatment with NG-hydroxy-L-arginine, an arginase inhibitor.


rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

Melichar B, Hu W, Patenia R, Melicharová K, Gallardo ST, Freedman R - J Transl Med (2003)

Cytostatic activity of IFN-γ, NG-hydroxy-L-arginine and the effect of polyamines on IFN-γ-induced cytostasis. NMP-1 cells were seeded at 104 cells/well into 96-well plates in RPMI 1640 with 10% fetal calf serum and cultured overnight. The next day, the cells were washed with phosphate buffered salines, and the medium was replaced with a low-arginine, serum-free medium (5% bovine serum albumin, 1.5% Iscove's modified Dulbecco's medium [GIBCO], 0.2% glucose, 0.01% magnesium sulfate, 0.016% calcium chloride, transferrin 5.5 μg/ml, and insulin 4 μg/ml in Hank's balanced salt medium [GIBCO], pH 7.4) alone or with rIFN-γ (500 U/ml). The arginine concentration of this medium was 6 μM, lower than plasma concentration (normal range, 50–126 μM [38]. After another 24 hours, rIFN-γ (500 U/ml), NG-hydroxy-L-arginine (250 μM), or rIFN-γ plus ornithine (1 mM), putrescine (1 mM), or spermidine (0.5 mM) was added. The cells were then pulsed with [3H] thymidine and incubated for another 24 hours. The cytostatic activity was determined as described in Materials and Methods. Similar results were observed with other EOC cell lines and when experiments were done with RPMI 1640 supplemented with 10% fetal calf serum. Data shown are representative of 3 separate experiments.
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Related In: Results  -  Collection

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Figure 3: Cytostatic activity of IFN-γ, NG-hydroxy-L-arginine and the effect of polyamines on IFN-γ-induced cytostasis. NMP-1 cells were seeded at 104 cells/well into 96-well plates in RPMI 1640 with 10% fetal calf serum and cultured overnight. The next day, the cells were washed with phosphate buffered salines, and the medium was replaced with a low-arginine, serum-free medium (5% bovine serum albumin, 1.5% Iscove's modified Dulbecco's medium [GIBCO], 0.2% glucose, 0.01% magnesium sulfate, 0.016% calcium chloride, transferrin 5.5 μg/ml, and insulin 4 μg/ml in Hank's balanced salt medium [GIBCO], pH 7.4) alone or with rIFN-γ (500 U/ml). The arginine concentration of this medium was 6 μM, lower than plasma concentration (normal range, 50–126 μM [38]. After another 24 hours, rIFN-γ (500 U/ml), NG-hydroxy-L-arginine (250 μM), or rIFN-γ plus ornithine (1 mM), putrescine (1 mM), or spermidine (0.5 mM) was added. The cells were then pulsed with [3H] thymidine and incubated for another 24 hours. The cytostatic activity was determined as described in Materials and Methods. Similar results were observed with other EOC cell lines and when experiments were done with RPMI 1640 supplemented with 10% fetal calf serum. Data shown are representative of 3 separate experiments.
Mentions: rIFN-γ produced significant growth inhibition in 7 of 8 cell lines examined, the exception being SKOV3, a cell line with negligible arginase activity (Table 1). Since the growth-inhibitory activity of rIFN-γ in certain EOC lines appeared to be accompanied with arginase inhibition, and since formation of ornithine from arginine catalyzed by arginase represents the first step in the synthesis of polyamines, we hypothesized the inhibition of arginase activity might be at least partly responsible for the growth-inhibitory effects of rIFN-γ on EOC cell lines. However, the addition of polyamines, ornithine, putrescine, or spermidine did not abolish the growth-inhibitory activity of rIFN-γ (Fig. 3). In addition, there was no interference with growth inhibition by rIFN-γ in EOC cell lines after treatment with NG-hydroxy-L-arginine, an arginase inhibitor.

Bottom Line: However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells.CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma.Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynecologic Oncology, The University of Texas, M,D, Anderson Cancer Center, Houston, Texas, U,S,A. rfreedma@mdanderson.org

ABSTRACT
BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

No MeSH data available.


Related in: MedlinePlus