scholarly journals Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF

2017 ◽  
Vol 24 (9) ◽  
pp. 459-474 ◽  
Author(s):  
W Imruetaicharoenchoke ◽  
A Fletcher ◽  
W Lu ◽  
R J Watkins ◽  
B Modasia ◽  
...  
Keyword(s):  
Colony Formation ◽  
Human Cancer ◽  
Soft Agar ◽  
Cellular Migration ◽  
3T3 Fibroblasts ◽  
Wide Range ◽  
Functional Consequences ◽  
Altered Cell ◽  
Proliferation Assays ◽  
Nih 3T3

Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer.

scholarly journals Transformation of NIH 3T3 fibroblasts by an activated form of p59hck.

1989 ◽  
Vol 9 (6) ◽  
pp. 2724-2727 ◽  
Author(s):  
S F Ziegler ◽  
S D Levin ◽  
R M Perlmutter
Keyword(s):  
Tyrosine Kinases ◽  
Soft Agar ◽  
Protein Tyrosine Kinases ◽  
3T3 Fibroblasts ◽  
Family Protein ◽  
Cloning Assay ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Soft Agar Cloning ◽  
Phenylalanine Residue

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

scholarly journals Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

1998 ◽  
Vol 18 (5) ◽  
pp. 2586-2595 ◽  
Author(s):  
Jaw-Ji Yang ◽  
Jong-Sun Kang ◽  
Robert S. Krauss
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Cyclin A ◽  
Soft Agar ◽  
Specific Cell ◽  
3T3 Cells ◽  
Anchorage Independent Growth ◽  
Nih 3T3 ◽  
Cell Cycle Machinery ◽  
Nih 3T3 Cells

ABSTRACT Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.

scholarly journals Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes.

1992 ◽  
Vol 12 (1) ◽  
pp. 190-197 ◽  
Author(s):  
S K Gupta ◽  
C Gallego ◽  
J M Lowndes ◽  
C M Pleiman ◽  
C Sable ◽  
...  
Keyword(s):  
Growth Regulation ◽  
Normal Growth ◽  
Soft Agar ◽  
Dominant Negative ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Signal Transduction Protein ◽  
Swiss 3T3 ◽  
Nih 3T3 ◽  
Mutant Polypeptides

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.

scholarly journals Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 807
Author(s):  
Hiu Yeung Lau ◽  
Jingyi Tang ◽  
Patrick J. Casey ◽  
Mei Wang
Keyword(s):  
Epithelial Cells ◽  
Colony Formation ◽  
Epithelial Mesenchymal Transition ◽  
Soft Agar ◽  
T Antigen ◽  
Adherent Cell ◽  
Functional Assay ◽  
Mesenchymal Transition ◽  
New Methods ◽  
Wide Range

The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

Transformation of NIH 3T3 fibroblasts by an activated form of p59hck

1989 ◽  
Vol 9 (6) ◽  
pp. 2724-2727
Author(s):  
S F Ziegler ◽  
S D Levin ◽  
R M Perlmutter
Keyword(s):  
Tyrosine Kinases ◽  
Soft Agar ◽  
Protein Tyrosine Kinases ◽  
3T3 Fibroblasts ◽  
Family Protein ◽  
Cloning Assay ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Soft Agar Cloning ◽  
Phenylalanine Residue

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes

1992 ◽  
Vol 12 (1) ◽  
pp. 190-197
Author(s):  
S K Gupta ◽  
C Gallego ◽  
J M Lowndes ◽  
C M Pleiman ◽  
C Sable ◽  
...  
Keyword(s):  
Growth Regulation ◽  
Normal Growth ◽  
Soft Agar ◽  
Dominant Negative ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Signal Transduction Protein ◽  
Swiss 3T3 ◽  
Nih 3T3 ◽  
Mutant Polypeptides

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.

Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts

1995 ◽  
Vol 74 (1) ◽  
pp. 207-217 ◽  
Author(s):  
T. Kanemasa ◽  
L. Gan ◽  
T. M. Perney ◽  
L. Y. Wang ◽  
L. K. Kaczmarek
Keyword(s):  
Protein Kinase ◽  
Action Potential ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
Delayed Rectifier ◽  
Time Pattern ◽  
3T3 Fibroblasts ◽  
Open Time ◽  
Wide Range ◽  
Nih 3T3

1. The Shaw-like voltage-activated potassium channel Kv3.1 is expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated that the Kv3.1 channel displays two gating behaviors, a short-open-time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.

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