p53 Mutants Have Selective Dominant-Negative Effects on Apoptosis but Not Growth Arrest in Human Cancer Cell Lines

ABSTRACT A bidirectional expression vector that allowed equal transcription of cloned wild-type and mutant p53 cDNAs from the same vector was developed. The vector was transfected into CaLu 6 lung carcinoma cells or Saos-2 osteosarcoma cells. All p53 mutants examined were recessive to wild-type p53 transactivation ofp21WAF1/CIP1 but dominant-negative for transactivation of Bax. An examination of effects on growth arrest and apoptotic pathways indicated that all mutants were recessive to wild type for growth arrest but only three of seven mutants were dominant negative for induction of apoptosis.

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Loss of mutant TP53 does not impair the sustained proliferation, survival or metastasis of diverse cancer cells

10.1101/2020.03.30.015263 ◽

2020 ◽

Author(s):

Zilu Wang ◽

François Vaillant ◽

Catherine Chang ◽

Chris Riffkin ◽

Elizabeth Lieschke ◽

...

Keyword(s):

Human Cancer ◽

Hot Spot ◽

Point Mutations ◽

Chemotherapeutic Agents ◽

Dominant Negative ◽

Loss Of Function ◽

Negative Effects ◽

Substantial Impact ◽

Human Cancer Cell Lines ◽

Cellular Stresses

AbstractThe tumour suppressor TP53 is the most frequently mutated gene in human cancer and these aberrations confer poor chemotherapeutic responses1-3. Point mutations typically cluster in the DNA binding domain, with certain ‘hot-spot’ residues disproportionally represented1-4 These mutations abrogate binding of the TP53 transcription factor to DNA and thereby prevent upregulation of genes critical for tumour suppression (loss-of-function)1-3. Mutant TP53 is reported to additionally contribute to tumour development, sustained growth and metastasis not only through dominant-negative effects on wild-type TP535 but also through neomorphic gain-of-function (GOF) activities6. Understanding the contributions of these postulated attributes of mutant TP53 to the development and expansion of tumours will facilitate the design of rational therapeutic strategies. Here we used CRISPR/CAS9 to delete mutant TP53 in a panel of diverse human cancer cell lines. The loss of mutant TP53 expression had no impact on the survival, proliferative capacity or metabolic state of the tumour cells, nor did it sensitise them to cellular stresses and chemotherapeutic agents. These data suggest that putative GOF effects of mutant TP53 are not universally required for the sustained survival and proliferation of fully malignant cells. Therefore, therapeutic approaches that abrogate expression or function of mutant TP53 would not be expected to have substantial impact.

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The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5725-5737.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5725-5737

Author(s):

M Au-Fliegner ◽

E Helmer ◽

J Casanova ◽

B M Raaka ◽

H H Samuels

Keyword(s):

Retinoic Acid ◽

Thyroid Hormone ◽

Dominant Negative ◽

Heptad Repeat ◽

Specific Response ◽

Wild Type ◽

Negative Effects ◽

Related Factors ◽

Response Element ◽

Response Elements

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

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The adenovirus E4orf4 protein induces growth arrest and mitotic catastrophe in H1299 human lung carcinoma cells

Oncogene ◽

10.1038/onc.2008.393 ◽

2008 ◽

Vol 28 (3) ◽

pp. 390-400 ◽

Cited By ~ 35

Author(s):

S Li ◽

A Szymborski ◽

M-J Miron ◽

R Marcellus ◽

O Binda ◽

...

Keyword(s):

Lung Carcinoma ◽

Human Lung ◽

Growth Arrest ◽

Carcinoma Cells ◽

Mitotic Catastrophe ◽

Lung Carcinoma Cells ◽

Human Lung Carcinoma

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Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

Journal of Bacteriology ◽

10.1128/jb.184.3.695-705.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 695-705 ◽

Cited By ~ 34

Author(s):

Joseph C. Chen ◽

Michael Minev ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Random Mutagenesis ◽

Dominant Negative ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Negative Effects ◽

Mutant Proteins ◽

Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

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Activation of peroxisome proliferator-activated receptor-γ stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells

Oncogene ◽

10.1038/sj.onc.1205279 ◽

2002 ◽

Vol 21 (14) ◽

pp. 2171-2180 ◽

Cited By ~ 82

Author(s):

Teturou Satoh ◽

Mitsuo Toyoda ◽

Hideki Hoshino ◽

Tsuyoshi Monden ◽

Masanabu Yamada ◽

...

Keyword(s):

Dna Damage ◽

Small Cell Lung Carcinoma ◽

Growth Arrest ◽

Small Cell ◽

Carcinoma Cells ◽

Peroxisome Proliferator ◽

Small Cell Lung ◽

Peroxisome Proliferator Activated Receptor ◽

Cell Lung Carcinoma ◽

Lung Carcinoma Cells

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Evidence for the multimeric structure of ferroportin

Blood ◽

10.1182/blood-2006-06-032516 ◽

2006 ◽

Vol 109 (5) ◽

pp. 2205-2209 ◽

Cited By ~ 47

Author(s):

Ivana De Domenico ◽

Diane McVey Ward ◽

Giovanni Musci ◽

Jerry Kaplan

Keyword(s):

Iron Overload ◽

Cultured Cells ◽

Dominant Negative ◽

Mouse Bone Marrow ◽

C6 Cells ◽

Wild Type ◽

Negative Effects ◽

Dominant Form ◽

Rat Glioma ◽

Glioma C6

Abstract Ferroportin (Fpn) (IREG1, SLC40A1, MTP1) is an iron transporter, and mutations in Fpn result in a genetically dominant form of iron overload disease. Previously, we demonstrated that Fpn is a multimer and that mutations in Fpn are dominant negative. Other studies have suggested that Fpn is not a multimer and that overexpression or epitope tags might affect the localization, topology, or multimerization of Fpn. We generated wild-type Fpn with 3 different epitopes, GFP, FLAG, and c-myc, and expressed these constructs in cultured cells. Co-expression of any 2 different epitope-tagged proteins in the same cell resulted in their quantitative coimmunoprecipitation. Treatment of Fpn-GFP/Fpn-FLAG–expressing cells with crosslinking reagents resulted in the crosslinking of Fpn-GFP and Fpn-FLAG. Western analysis of rat glioma C6 cells or mouse bone marrow macrophages exposed to crosslinking reagents showed that endogenous Fpn is a dimer. These results support the hypothesis that the dominant inheritance of Fpn–iron overload disease is due to the dominant-negative effects of mutant Fpn proteins.

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11 Study of Polynucleotide Kinase/Phosphatase (PNKP) Mutations Found in a Patient with Microcephaly, Seizures, and Developmental Delay (MCSZ) and Glioblastoma

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2018.257 ◽

2018 ◽

Vol 45 (S3) ◽

pp. S2-S2

Author(s):

Bingcheng Jiang ◽

Chibawanye I. Ene ◽

Bonnie Cole ◽

Jeff Ojemann ◽

Sarah Leary ◽

...

Keyword(s):

Human Cancer ◽

Neurodevelopmental Disorder ◽

Site Directed Mutagenesis ◽

Expression Vectors ◽

Wild Type ◽

Double Mutation ◽

Mutant Proteins ◽

Human Cancer Cell Lines ◽

Mammalian Expression ◽

Polynucleotide Kinase

The enzyme polynucleotide kinase/phosphatase (PNKP) plays a key role in DNA repair by resolving the chemistry at DNA strand breaks. Mutations in PNKP (chromosome 19q13.4) are known to cause MCSZ, a serious neurodevelopmental disorder, but to date there has been no link to cancer initiation or progression. However, a child with MCSZ recently presented at Seattle Children's Hospital with a 3-cm glioblastoma. The child was shown to have two germline mutations in PNKP. To study the effects of the PNKP mutations found in this patient, we generated mutant PNKP cDNAs carrying either the individual mutations or the double mutation using site directed mutagenesis. These cDNAs were incorporated into bacterial and mammalian expression vectors. The bacterially expressed mutant proteins as well as the wild type have been purified and are undergoing testing for PNKP DNA kinase and phosphatase activity. The PNKP cDNAs, fused to GFP, were expressed in Hela and HCT116 human cancer cell lines. High-content analysis and micro-irradiation techniques are being used to determine PNKP localization within the cells and recruitment to damaged DNA. Our preliminary results indicate that the mutations alter the ratio of nuclear to cytoplasmic PNKP compared to the wild-type protein.

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