Colletofragarone A2 and Colletoins A–C from a Fungus Colletotrichum sp. Decrease Mutant p53 Levels in Cells

Abstract Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32C and mutant p53 phenotype at 37C (Akay et al. Akay et al., AACR; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation, 2005). Employing the Affymetrix Microarray technology to compare global gene expression in myeloma cell lines we identified a number of new genes affected by ATO (Gazitt et al. ASH presentation, 2005). One of these genes was heat shock protein 90 (HSP90). We therefore hypothesized that treatment of myeloma cells with blockers of HSP90 such as geldanamycin or its newly discovered potent derivative; 17-allylamino-17-demethoxygeldanamycin (17-AAG) in combination with ATO will result with synergy in the induction of apoptosis in these cells. Indeed, treatment of IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) with 17 AAG (0 to 3uM) resulted with a time/dose induced apoptosis to a maximum of 25% apoptosis by annexin V. Treatment with ATO alone at 2.5 uM resulted with 22% apoptosis following 24 hours of treatment. However, ATO synergized with 17 AAD to induce 2–3 fold higher apoptosis compared to the sum of the individual drugs in each dose tested. In contrast, only additive effect was observed between 17 AAG and ATO in the induction of mitochondrial membrane (MM) depolarization as measured by TMRE fluorescence and in the depletion of glutathione measured by MCB fluorescence. Interestingly, 17 AAG did not have any effect on generation of reactive oxygen species as measured by DHR fluorescence. Finally, 17 AAG induced mild arrest of cells at G2/M with marked increase in G2/M arrest when combined with ATO, which by itself did not increase the percentage of cells at G2/M. Similar trend was observed in U266 cells, in which apoptosis, MM depolarization, depletion of glutathione and G2/M arrest were much higher with 17 AAG alone or with ATO alone, and hence only additive effect was observed between the 2 drugs, at the same dosing used for the IM9 cells. Western immunoblot analysis of the levels of HSP 90 -alpha (90kd) and beta (81kd) subunits revealed slight inhibition with 17 AAG alone and ATO alone with marked decreased in HSP90 inhibition observed in cells treated with both drugs. Furthermore, analysis of the proteins involved in the intrinsic and extrinsic apoptosic pathways revealed a shift from activation of the intrinsic to activation of the extrinsic apoptotic pathway in IM9 cells treated with the combination of 17 AAG and ATO, similar to the pattern observed in U266 treated with ATO alone. These results strongly suggest that 17 AAG could potentiate the effect of ATO, in myeloma patients treated with the combination of the 2 drugs.

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Arsenic Trioxide Induced p53-Dependant Apoptotic Signals in a Temperature Sensitive (Ts) p53 Mutant, BRK Cell Line.

Blood ◽

10.1182/blood.v104.11.4318.4318 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4318-4318

Author(s):

Cagla Akay ◽

Yair Gazitt

Keyword(s):

Arsenic Trioxide ◽

Mitochondrial Membrane ◽

Temperature Sensitive ◽

G1 Arrest ◽

Mutant P53 ◽

Apoptotic Pathway ◽

Apoptotic Pathways ◽

Induced Apoptosis ◽

Extrinsic Apoptotic Pathways ◽

In Cells

Abstract Mutations in the p53 gene are the most common genetic abnormality in human cancers and are the cause for drug resistance. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53 whereas in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. To further establish the differential effect of ATO in relation to the p53 status we studied the effect of temperature shift in temperature sensitive (Ts)-p53 expressing baby rat kidney (BRK) cells. We studied by Western immunoblotting the activation of the intrinsic and the extrinsic apoptotic pathways in ATO-induced apoptosis of BRK cells cultured at 32°C (w.t. p53 phenotype) and 37°C (mutant p53 phenotype). As expected, at 32°C, we observed a G1 arrest through activation of p21. We also observed depolarization of mitochondrial membrane; the release of cytochrome C and activation of caspase-9 and apoptosis as measured by Annexin V. We also observed release of SMAC at 32°C. In contrast, at 37°C we observed a G2/M arrest with no activation of p21 with activation of the extrinsic apoptotic pathway through early induction of TRAIL and TRAIL receptor- R2, activation of caspase-8, activation of BID, degradation of FLIP, rapid depolarization of mitochondrial membrane and release of AIF from mitochondria to the cytosol. We also demonstrate by flow cytometry and confocal imaging translocation of AIF to the nucleus in ATO-induced apoptosis at 37°C but not at 32°C. These results further substantiate our p53 model (Akay and Gazitt, Cell Cycle2:258–268, 2003).

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The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.18.10355 ◽

1999 ◽

Vol 96 (18) ◽

pp. 10355-10360 ◽

Cited By ~ 58

Author(s):

C. Gobert ◽

A. Skladanowski ◽

A. K. Larsen

Keyword(s):

Topoisomerase I ◽

Mutant P53 ◽

Dna Topoisomerase I ◽

Wild Type ◽

Dna Topoisomerase ◽

In Cells

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Effect of p53 Silencing on Asenic Tioxide-Induced Apoptotic Pathways in a Myeloma Cell Line Expressing w.t.p53.

Blood ◽

10.1182/blood.v106.11.4387.4387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4387-4387

Author(s):

Kircelli Fatih ◽

Akay Cagla ◽

Gazitt Yair

Keyword(s):

Cell Line ◽

Target Genes ◽

G1 Arrest ◽

Mutant P53 ◽

Apoptotic Pathway ◽

Potent Inducer ◽

Phase Ii Studies ◽

Mitochondrial Membrane Depolarization ◽

Apoptotic Pathways ◽

In Cells

Abstract Mutations in the p53 gene are the most common genetic abnormality in human cancers. P53 is a tumor suppressor gene, which regulates a wide variety of target genes controlling cell cycle and apoptosis. Arsenic Trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. Our finding in this respect were further supported by our recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al.,AACR; Abstract #5344, 2005). We hypothesized that knocking out of p53 or p21 mRNA will result in modifying ATO-induced apoptotic pathways as observed using the p53-Ts conditional mutant of BRK cells. Thus, IM9 cells (expressing w.t.p53) were transfected [AMAXA Nucleoporator, ~60% transfection by green fluorescence protein (GFP) tag] with a GFP-SiRNA expressing plasmid co-expressing anti-sense RNA sequences for p53 (GFP/Sip53) and p21 (GFP/Sip21). As a control, GFP expressing plasmid not containing SiRNA (GFP/cont) was used. We observed in GFP/cont-transfected cells a G1 arrest through activation of p21 and moderate apoptosis, primarily through activation of procaspase 9, with no activation of BID and procaspase 8. In cells transfected with GFP/Sip53 or GFP/Sip21 plasmids we observed rapid mitochondrial membrane depolarization and apoptosis primarily through activation of the extrinsic apoptotic pathway with downregulation of FLIP, activation of procaspase 8 and BID, enhanced release of AIF from mitochondria and rapid apoptosis. Depletion of glutathione was observed with or without silencing of p21 or p53. Furthermore, silencing of p53 or p21 resulted with a decrease in ATO-induced activation of c-jun and Erk and in a decrease in the activation of Akt. These results further substantiate the model we put forward for the role of p53 in ATO-induced apoptosis in myeloma and other cancer cell lines.

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A mutant p53 protein is required for maintenance of the transformed phenotype in cells transformed with p53 plus ras cDNAs.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.9.3952 ◽

1992 ◽

Vol 89 (9) ◽

pp. 3952-3956 ◽

Cited By ~ 30

Author(s):

G. P. Zambetti ◽

D. Olson ◽

M. Labow ◽

A. J. Levine

Keyword(s):

P53 Protein ◽

Mutant P53 ◽

In Cells

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Reduced levels of α1 (I) collagen mRNA in cells immortalized by mutant p53 or transformed by ras

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(91)90209-5 ◽

1991 ◽

Vol 1129 (1) ◽

pp. 34-42 ◽

Cited By ~ 4

Author(s):

Orly Eizenberg ◽

Moshe Oren

Keyword(s):

Mutant P53 ◽

Collagen Mrna ◽

In Cells

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Increased p53 site-specific DNA binding in cells producing mutant p53

Cancer Letters ◽

10.1016/0304-3835(95)03936-q ◽

1995 ◽

Vol 96 (2) ◽

pp. 225-231 ◽

Cited By ~ 2

Author(s):

C. Dees ◽

C.C. Travis

Keyword(s):

Dna Binding ◽

Mutant P53 ◽

Site Specific ◽

In Cells

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Global Gene Expression Changes during Arsenic Trioxide -Induced Apoptosis in Myeloma Cell Lines Expressing Mutant or w.t p53.

Blood ◽

10.1182/blood.v106.11.4351.4351 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4351-4351

Author(s):

Yair Gazitt ◽

Cagla Akay ◽

Fatih Kircelli

Keyword(s):

Gene Expression ◽

Arsenic Trioxide ◽

Global Gene Expression ◽

Cyclin D3 ◽

Mutant P53 ◽

Affymetrix Microarray ◽

Potent Inducer ◽

Myeloma Cells ◽

New Genes ◽

In Cells

Abstract Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia and is being tested in phase II studies in various types of hematological malignancies and solid tumors. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging primarily the intrinsic apoptotic pathway in cells expressing w.t. p53. In contrast, in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. These findings were further supported by a recent study using a p53 temperature sensitive (p53Ts) mutant cell line, BRK, expressing w.t. p53 at 32°C and mutant p53 phenotype at 37°C (Akay et al. Akay et al., AACR; Abstract #5344, 2005). Furthermore, myeloma cells expressing w.t. p53 transfected with SiRNA for p53 or p21 behaved like cells with mutant p53 (Kircelli et al. ASH presentation,2005). In order to identify new genes affected by ATO we used the Affymetrix Microarray technology to compare global gene expression in IM9 myeloma cells (w.t. p53) and U266 myeloma cells (mutant p53) following 0, 1,5 and 10 hours of treatment with ATO. We found ≥2 fold increase in gene expression by Affymetrix Microarray Suite Software (MAS) in 94 genes at 5 h and 455 genes at 10 h with an increase in 134, at both 5 and 10 h of treatment. By similar analysis, 263 genes were decreased at 5 h and 679 genes were decreased at 10 h with 204 genes decreased at both time points. Similar analysis with GeneSpring (GS) software revealed an increase in 202 genes and a decrease in 233 genes at both 5 and 10 h. Combination of the 2 analysis methods yielded 90 consistent increasers and 64 consistent decreasers. A great number of these genes in whom we detected changes in this study are genes that were previously identified by us and by others using Western immunoblotting. In addition, we observed differential effect of ATO in IM9 and U266 myeloma cells in apoptosis-related genes (HRK, BID, MCL1); cell cycle-related genes (GADD45, Cyclin D1, cyclin D2 and cyclin D3); signal transduction proteins (ERK, NFkB, ATM, ATR, CHK2, TRAILR2, TNF-R5/6 and VEGF); in chaperon proteins; cyclophyllin B; SAT, ToPoIIA and others. Systematic validation of these changes on the protein levels is ongoing.

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Ninjurin 1 has two opposing functions in tumorigenesis in a p53-dependent manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711814114 ◽

2017 ◽

Vol 114 (43) ◽

pp. 11500-11505 ◽

Cited By ~ 12

Author(s):

Hee Jung Yang ◽

Jin Zhang ◽

Wensheng Yan ◽

Seong-Jun Cho ◽

Christopher Lucchesi ◽

...

Keyword(s):

Cell Growth ◽

Inflammatory Diseases ◽

Biological Significance ◽

Mrna Translation ◽

Mutant P53 ◽

Dependent Manner ◽

Cell Growth And Migration ◽

P53 Expression ◽

And Migration ◽

In Cells

WT p53 is critical for tumor suppression, whereas mutant p53 promotes tumor progression. Nerve injury-induced protein 1 (Ninj1) is a target of p53 and forms a feedback loop with p53 by repressing p53 mRNA translation. Here, we show that loss of Ninj1 increased mutant p53 expression and, subsequently, enhanced cell growth and migration in cells carrying a mutant p53. In contrast, loss of Ninj1 inhibited cell growth and migration in cells carrying a WT p53. To explore the biological significance of Ninj1, we generated a cohort of Ninj1-deficient mice and found that Ninj1+/− mice were prone to systemic inflammation and insulitis, but not to spontaneous tumors. We also found that loss of Ninj1 altered the tumor susceptibility in both mutant p53 and p53-null background. Specifically, in a mutant p53(R270H) background, Ninj1 deficiency shortened the lifespan, altered the tumor spectrum, and increased tumor burden, likely via enhanced expression of mutant p53. In a p53-null background, Ninj1 deficiency significantly increased the incidence of T-lymphoblastic lymphoma. Taken together, our data suggest that depending on p53 genetic status, Ninj1 has two opposing functions in tumorigenesis and that the Ninj1–p53 loop may be targeted to manage inflammatory diseases and cancer.

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