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Expression of MTAP inhibits tumor-related phenotypes in HT1080 cells via a mechanism unrelated to its enzymatic function.

Tang B, Kadariya Y, Chen Y, Slifker M, Kruger WD - G3 (Bethesda) (2014)

Bottom Line: Treatment of MTAP-expressing cells with a potent inhibitor of MTAP's enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP- phenotype.To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes.Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.

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Related in: MedlinePlus

MTAP expression and activity in HT1080 cells. (A) Western blot showing levels of MTAP in extracts from stably transfected HT1080 cells. M− is the parent cell line transfected with vector alone (pTRE2). M+ has been transfected with the MTAP expressing construct pTRE2:MTAP. M+I is the identical to M+, except the cells have been treated for 72 hr with 10 μM the MTAP inhibitor, MT-DAD-Me-ImmA. Hela contains extract from a MTAP+ Hela cell. D220A contains extract from a HT1080 cell that has been transfected with a plasmid that expresses D220A MTAP (pTRE2:MTAP:D220A). (B) MTAP enzymatic activity measured in the same extracts as used in (A). Error bars show SD of enzyme assay (n = 4). All means are different from each other as assessed by one-way ANOVA followed by Tukey test (P < 0.01 for all comparisons).
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fig1: MTAP expression and activity in HT1080 cells. (A) Western blot showing levels of MTAP in extracts from stably transfected HT1080 cells. M− is the parent cell line transfected with vector alone (pTRE2). M+ has been transfected with the MTAP expressing construct pTRE2:MTAP. M+I is the identical to M+, except the cells have been treated for 72 hr with 10 μM the MTAP inhibitor, MT-DAD-Me-ImmA. Hela contains extract from a MTAP+ Hela cell. D220A contains extract from a HT1080 cell that has been transfected with a plasmid that expresses D220A MTAP (pTRE2:MTAP:D220A). (B) MTAP enzymatic activity measured in the same extracts as used in (A). Error bars show SD of enzyme assay (n = 4). All means are different from each other as assessed by one-way ANOVA followed by Tukey test (P < 0.01 for all comparisons).

Mentions: HT1080 cells are an immortalized human fibrosarcoma cell line that lacks MTAP expression (Tang et al. 2000). We created MTAP+ (M+) and MTAP− (M−) HT1080 cell lines by stably transfecting either a MTAP expression plasmid or an empty vector control and pooled multiple clones together to minimize the effects of integration events (Tang et al. 2012). The amount of MTAP protein and activity in M+ cells were slightly reduced from those observed in Hela cells (Figure 1, A and B.; compare lane M+ with Hela), indicating that MTAP is being expressed in these cells at near physiologic levels. (We will discuss lanes labeled M+I and D220A in the last two sections.)


Expression of MTAP inhibits tumor-related phenotypes in HT1080 cells via a mechanism unrelated to its enzymatic function.

Tang B, Kadariya Y, Chen Y, Slifker M, Kruger WD - G3 (Bethesda) (2014)

MTAP expression and activity in HT1080 cells. (A) Western blot showing levels of MTAP in extracts from stably transfected HT1080 cells. M− is the parent cell line transfected with vector alone (pTRE2). M+ has been transfected with the MTAP expressing construct pTRE2:MTAP. M+I is the identical to M+, except the cells have been treated for 72 hr with 10 μM the MTAP inhibitor, MT-DAD-Me-ImmA. Hela contains extract from a MTAP+ Hela cell. D220A contains extract from a HT1080 cell that has been transfected with a plasmid that expresses D220A MTAP (pTRE2:MTAP:D220A). (B) MTAP enzymatic activity measured in the same extracts as used in (A). Error bars show SD of enzyme assay (n = 4). All means are different from each other as assessed by one-way ANOVA followed by Tukey test (P < 0.01 for all comparisons).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4291467&req=5

fig1: MTAP expression and activity in HT1080 cells. (A) Western blot showing levels of MTAP in extracts from stably transfected HT1080 cells. M− is the parent cell line transfected with vector alone (pTRE2). M+ has been transfected with the MTAP expressing construct pTRE2:MTAP. M+I is the identical to M+, except the cells have been treated for 72 hr with 10 μM the MTAP inhibitor, MT-DAD-Me-ImmA. Hela contains extract from a MTAP+ Hela cell. D220A contains extract from a HT1080 cell that has been transfected with a plasmid that expresses D220A MTAP (pTRE2:MTAP:D220A). (B) MTAP enzymatic activity measured in the same extracts as used in (A). Error bars show SD of enzyme assay (n = 4). All means are different from each other as assessed by one-way ANOVA followed by Tukey test (P < 0.01 for all comparisons).
Mentions: HT1080 cells are an immortalized human fibrosarcoma cell line that lacks MTAP expression (Tang et al. 2000). We created MTAP+ (M+) and MTAP− (M−) HT1080 cell lines by stably transfecting either a MTAP expression plasmid or an empty vector control and pooled multiple clones together to minimize the effects of integration events (Tang et al. 2012). The amount of MTAP protein and activity in M+ cells were slightly reduced from those observed in Hela cells (Figure 1, A and B.; compare lane M+ with Hela), indicating that MTAP is being expressed in these cells at near physiologic levels. (We will discuss lanes labeled M+I and D220A in the last two sections.)

Bottom Line: Treatment of MTAP-expressing cells with a potent inhibitor of MTAP's enzymatic activity (MT-DADMe-ImmA) did not result in a MTAP- phenotype.To confirm this, we introduced a catalytically inactive version of MTAP, D220A, into HT1080 cells and found that this mutant was fully capable of reversing the soft agar colony formation, migration, and matrix metalloproteinase phenotypes.Our results show that MTAP affects cellular phenotypes in HT1080 cells in a manner that is independent of its known enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.

Show MeSH
Related in: MedlinePlus