Development of a quantitative immunofluorescent method for analysis of nuclear and cytoplasmic p53 and p21 in circulating tumor cells (CTC) as biomarkers of response to p53-targeted therapy.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13575-e13575
Author(s):  
Jacky Woo ◽  
Weiguo Wu ◽  
Vladislava O. Melnikova ◽  
Kenna Lynn Anderes ◽  
Darren W. Davis
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cell Lines ◽  
Laser Scanning ◽  
Checkpoint Inhibitors ◽  
A549 Cells ◽  
Cell Cycle Checkpoint ◽  
P53 Expression ◽  
P53 Status

e13575 Background: The tumor suppressor p53 is involved in many aspects of cell cycle control and p53 mutations are the most common genetic abnormality in solid tumors. Several diverse approaches targeting p53 signaling including activators of p53, cell cycle checkpoint inhibitors and MdM2 inhibitors are under development and will require determination of p53 status to demonstrate proof of concept and ultimately drive patient selection. Measurement of p53 status in tumor biopsies is often hindered by limited availability of tissues. Detection of p53 status in CTCs offers an alternative to biopsy. Methods: A multiplex immunofluorescent (IF) method for identification of CTCs was developed using cytokeratin, CD45 and DAPI. DO7, a p53 specific antibody, and total and phospho-p21 antibodies were optimized for multiplex detection. Bleomycin (50 mU/mL) was used to activate p53 in A549 p53 wt cells, HT29 and SkBr3 p53 mutant cells. Single cell-based IF analysis was performed pre and post treatment by quantitative Laser Scanning Cytometry (LSC). Results: Wild type A549 cells expressed lower baseline levels of nuclear p53 compared to mutant p53 cell lines HT29 and SKBr3. Total and phosphorylated p21 was also lower in A549 cells compared to HT29 and SKBr3 cells. Bleomycin treatment of wt A549 cells led to a transient increase in p53 expression by 30% while mutant HT29 and SKBr3 cells did not show any change in p53. Treatment of A549 cells with bleomycin led to a 100% increase in total p21and a 70% increase in phosphorylated p21. No change in p21 was observed in p53 mutant HT29 and SKBr3 cell lines. Conclusions: We developed a sensitive, quantitative method for detection of nuclear and cytoplasmic p53 and p21 protein in cell lines that functionally discriminates between p53 wt and p53 mutant status. The method is transferable to circulating tumor cells (CTCs) and may have utility in selecting patients based on functional p53 status and predicting responses to p53 pathway targeted agents.

scholarly journals The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6565
Author(s):  
Jennifer H. Foster ◽  
Eveline Barbieri ◽  
Linna Zhang ◽  
Kathleen A. Scorsone ◽  
Myrthala Moreno-Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Tumor Weight ◽  
Cycle Arrest ◽  
P53 Status ◽  
Neuroblastoma Cell Lines ◽  
Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

scholarly journals Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

2021 ◽  
Vol 14 (7) ◽  
pp. 682
Author(s):  
Jianling Bi ◽  
Garima Dixit ◽  
Yuping Zhang ◽  
Eric J. Devor ◽  
Haley A. Losh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endometrial Cancer ◽  
Tyrosine Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Cell Cycle Checkpoint ◽  
Mitotic Catastrophe ◽  
M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

scholarly journals Histone Deacetylase Inhibitors Trigger a G2 Checkpoint in Normal Cells That Is Defective in Tumor Cells

2000 ◽  
Vol 11 (6) ◽  
pp. 2069-2083 ◽  
Author(s):  
Ling Qiu ◽  
Andrew Burgess ◽  
David P. Fairlie ◽  
Helen Leonard ◽  
Peter G. Parsons ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Histone Deacetylase ◽  
Cell Cycle Progression ◽  
Histone Deacetylase Inhibitors ◽  
Cell Cycle Checkpoint ◽  
Phase Cell ◽  
Selective Toxicity ◽  
Protective Mechanisms ◽  
G2 Checkpoint

Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity. An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers. Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines. Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death. This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states. Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.

Differential radiosensitisation of bladder cancer cell lines by gemcitabine and its relationship to P53 status and cell cycle perturbations

2002 ◽  
Vol 1 (1) ◽  
pp. 42
Author(s):  
Vijay Sangar ◽  
Richard Cowan ◽  
Steve Roberts ◽  
Geoff Margison ◽  
Jolyon Hendry ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bladder Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Bladder Cancer Cell ◽  
P53 Status

scholarly journals Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals

2013 ◽  
Vol 45 (19) ◽  
pp. 907-916
Author(s):  
Liwen Zhang ◽  
Dennis A. Simpson ◽  
Cynthia L. Innes ◽  
Jeff Chou ◽  
Pierre R. Bushel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Checkpoint ◽  
G2 Checkpoint ◽  
P53 Signaling ◽  
Normal Individuals ◽  
Gene Expression Signatures ◽  
Cycle Checkpoint ◽  
Atm Cell

Ataxia telangiectasia (AT) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia-mutated gene ( ATM). AT carriers with one mutant ATM allele are usually not severely affected although they carry an increased risk of developing cancer. There has not been an easy and reliable diagnostic method to identify AT carriers. Cell cycle checkpoint functions upon ionizing radiation (IR)-induced DNA damage and gene expression signatures were analyzed in the current study to test for differential responses in human lymphoblastoid cell lines with different ATM genotypes. While both dose- and time-dependent G1 and G2 checkpoint functions were highly attenuated in ATM−/− cell lines, these functions were preserved in ATM+/− cell lines equivalent to ATM+/+ cell lines. However, gene expression signatures at both baseline (consisting of 203 probes) and post-IR treatment (consisting of 126 probes) were able to distinguish ATM+/− cell lines from ATM+/+ and ATM−/− cell lines. Gene ontology (GO) and pathway analysis of the genes in the baseline signature indicate that ATM function-related categories, DNA metabolism, cell cycle, cell death control, and the p53 signaling pathway, were overrepresented. The same analyses of the genes in the IR-responsive signature revealed that biological categories including response to DNA damage stimulus, p53 signaling, and cell cycle pathways were overrepresented, which again confirmed involvement of ATM functions. The results indicate that AT carriers who have unaffected G1 and G2 checkpoint functions can be distinguished from normal individuals and AT patients by expression signatures of genes related to ATM functions.

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