scholarly journals Dynamic variability in apoptotic threshold as a strategy for combating fractional killing

2018 ◽  
Author(s):  
Baohua Qiu ◽  
Jiajun Zhang ◽  
Tianshou Zhou
Keyword(s):  
Cancer Cells ◽  
First Passage Time ◽  
P53 Protein ◽  
P53 Gene ◽  
Threshold Level ◽  
Passage Time ◽  
Genetic Mutation ◽  
P53 Expression ◽  
Apoptosis Inducing Agents ◽  
Cell Variation

AbstractFractional killing, which is a significant impediment to successful chemotherapy, is observed even in a population of genetically identical cancer cells exposed to apoptosis-inducing agents. This phenomenon arises not from genetic mutation but from cell-to-cell variation in the activation timing and level of the proteins that regulate apoptosis. To understand the mechanism behind the phenomenon, we formulate complex fractional killing processes as a first-passage time (FPT) problem with a stochastically fluctuating boundary. Analytical calculations are performed for the FPT distribution in a toy model of stochastic p53 gene expression, where the cancer cell is killed only when the p53 expression level crosses an activity apoptotic threshold. Counterintuitively, we find that threshold fluctuations can effectively enhance cellular killing by significantly decreasing the mean time that the p53 protein reaches the threshold level for the first time. Moreover, faster fluctuations lead to the killing of more cells. These qualitative results imply that dynamic variability in threshold is an unneglectable stochastic source, and can be taken as a strategy for combating fractional killing of cancer cells.

scholarly journals Stochastic fluctuations in apoptotic threshold of tumour cells can enhance apoptosis and combat fractional killing

2020 ◽  
Vol 7 (2) ◽  
pp. 190462 ◽  
Author(s):  
Baohua Qiu ◽  
Tianshou Zhou ◽  
Jiajun Zhang
Keyword(s):  
Cancer Cells ◽  
First Passage Time ◽  
P53 Protein ◽  
P53 Gene ◽  
Threshold Level ◽  
Passage Time ◽  
Genetic Mutation ◽  
P53 Expression ◽  
Stochastic Fluctuations ◽  
Apoptosis Inducing Agents

Fractional killing, which is a significant impediment to successful chemotherapy, is observed even in a population of genetically identical cancer cells exposed to apoptosis-inducing agents. This phenomenon arises not from genetic mutation but from cell-to-cell variation in the activation timing and level of the proteins that regulates apoptosis. To understand the mechanism behind the phenomenon, we formulate complex fractional killing processes as a first-passage time (FPT) problem with a stochastically fluctuating boundary. Analytical calculations are performed for the FPT distribution in a toy model of stochastic p53 gene expression, where the cancer cell is killed only when the p53 expression level crosses an active apoptotic threshold. Counterintuitively, we find that threshold fluctuations can effectively enhance cellular killing by significantly decreasing the mean time that the p53 protein reaches the threshold level for the first time. Moreover, faster fluctuations lead to the killing of more cells. These qualitative results imply that fluctuations in threshold are a non-negligible stochastic source, and can be taken as a strategy for combating fractional killing of cancer cells.

scholarly journals Analysis of stochastic timing of intracellular events with gene switching

2019 ◽  
Author(s):  
Khem Raj Ghusinga ◽  
Abhyudai Singh
Keyword(s):  
First Passage Time ◽  
Regulatory Protein ◽  
Gene Activation ◽  
Threshold Level ◽  
Passage Time ◽  
First Passage ◽  
Cellular Processes ◽  
Event Timing ◽  
Activation Rates ◽  
Gene Switching

AbstractAn important step in execution of several cellular processes is accumulation of a regulatory protein up to a specific threshold level. Since production of a protein is inherently stochastic, the time at which its level crosses a threshold exhibits cell-to-cell variation. A problem of interest is to characterize how the statistics of event timing is affected by various steps of protein expression. Our previous work studied this problem by considering a gene expression model where gene was always active. Here we extend our analysis to a scenario where gene stochastically switches between active and inactive states. We formulate event timing as the first-passage time for a protein’s level to cross a threshold and investigate how the rates of gene activation/inactivation affect the distribution and moments of the first-passage time. Our results show that both the time-scale of gene switching with respect to the protein degradation rate as well as the ratio of the gene inactivation to gene activation rates are important parameters in shaping the event-timing distribution.

scholarly journals Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

2019 ◽  
Vol 9 (23) ◽  
pp. 5170 ◽  
Author(s):  
G.M. Azevedo ◽  
J.F.A. Valente ◽  
A. Sousa ◽  
A.Q. Pedro ◽  
P. Pereira ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Plasmid Dna ◽  
P53 Protein ◽  
Metabolic Stress ◽  
Suppressor Gene ◽  
Experimental Conditions ◽  
P53 Expression ◽  
Supercoiled Plasmid ◽  
Protein P53

The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.

scholarly journals Mutation of the p53 gene in human acute myelogenous leukemia

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1500-1507 ◽  
Author(s):  
JM Slingerland ◽  
MD Minden ◽  
S Benchimol
Keyword(s):  
Acute Myelogenous Leukemia ◽  
P53 Protein ◽  
Point Mutations ◽  
P53 Gene ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
P53 Expression ◽  
Coding Sequence ◽  
Acute Myelogenous ◽  
Blast Cells

Abstract Heterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t 1/2). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t 1/2 and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t 1/2 of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

scholarly journals Overexpression of wild-type p53 gene renders MCF-7 breast cancer cells more sensitive to the antiproliferative effect of progesterone

2003 ◽  
Vol 179 (1) ◽  
pp. 55-62 ◽  
Author(s):  
M Alkhalaf ◽  
AM El-Mowafy
Keyword(s):  
Breast Cancer ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
P53 Protein ◽  
P53 Gene ◽  
Inhibitory Effect ◽  
Antiproliferative Effect ◽  
Mcf 7

We have recently shown that growth inhibition of breast cancer cells by progesterone is due to the induction of cell differentiation, but not apoptosis. Because the tumor suppressor protein p53 plays a central role in normal cell growth and in tumor suppression, we have examined the effect of progesterone on the levels of this protein in MCF-7 cells. We show here that the antiproliferative effect of progesterone is accompanied with down-regulation of endogenous p53 protein. To study the effect of progesterone on cell growth in the presence of normal levels of p53 protein, we used transient transfection to overexpress p53 protein. MCF-7 cells were transfected with a p53 expressing vector that contains p53 human cDNA under the control of a cytomegalovirus promoter. Cell growth, cell viability, and apoptosis were analyzed in the transfected cells after six days of exposure to 100 nM progesterone. We show here that progesterone significantly enhances growth inhibition and apoptosis in MCF-7 cells overexpressing p53, but not in cells transfected with the control vector. These data suggest that re-establishing p53 function in MCF-7 breast cancer cells renders them more sensitive to the growth inhibitory effect of progesterone.

scholarly journals Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

1985 ◽  
Vol 5 (1) ◽  
pp. 127-132 ◽  
Author(s):  
D Wolf ◽  
N Harris ◽  
N Goldfinger ◽  
V Rotter
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cells ◽  
Cdna Clone ◽  
P53 Protein ◽  
P53 Gene ◽  
Full Length ◽  
Antigenic Determinants ◽  
Entire Group ◽  
P53 Expression ◽  
P53 Antibodies

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.

Effects of Bcl-2, Bax and P53 Gene Protein Expressions in Gastric Cancer Tissue on the Apoptosis of Cancer Cells

2013 ◽  
Vol 423-426 ◽  
pp. 358-361
Author(s):  
Lei Zhang ◽  
Da Peng Li ◽  
Guan Nan Lu
Keyword(s):  
Gastric Cancer ◽  
P53 Protein ◽  
P53 Gene ◽  
Gastric Cancer Tissue ◽  
Normal Lung ◽  
Cancer Tissue ◽  
P53 Expression ◽  
Bax Protein ◽  
Significant Difference ◽  
Positive Rate

Objective: To investigate the expression of p53 gene in gastric cancer tissue and its correlation with bcl-2 and bax protein expression in relationship and discuss the significance of their correlation in clinic. Methods: Pathological specimens from 100 gastric cancer patients with complete medical data and 24 normal lung tissue specimens were selected. Immunohistochemical streptavidin-biotin-peroxidase assay was used to detect the expression of bcl-2, bax and p53 protein. Results: p53 expression was positively correlated with bcl-2 expression (Pearson's R =0.491, P <0.05). The positive expression of p53 was no significantly correlated with bax expression (P> 0.05). The positive rate of p53 expression in the well-differentiated gastric cancer tissues was 17.6%, the positive rate in the differentiated tissues was 90.9%, the positive rate in the poorly differentiated tissues was 72.7%, and There was a significant difference in the expression of p53 in the three tissues (P <0.05). The expression of p53 in the TNM staging showed that the positive rate in stage Iand IIwas 27.3%, the positive rate in stage III and stage IVwas 89.3%, the difference between them was significant (P <0.05). Conclusions: The expression of p53 gene has an obvious clinical significance for the assessment of degree of malignancy and the prognosis of gastric cancer. Bcl-2 and p53 gene expression could jointly promote the development of gastric cancer in a synergistic way by acting on different stages of apoptosis, or bcl-2s participating in p53-induced mitochondria-mediated apoptosis signaling pathways.

scholarly journals Correlation between p53 expression and hepatocellular carcinoma differentiation grade

2020 ◽  
Vol 55 (1) ◽  
pp. 25-27
Author(s):  
B. K. ISSAMATOV ◽  
E. A. YENIN ◽  
U. Sh. MEDEUBEKOV ◽  
B. B. BAYMAKHANOV
Keyword(s):  
Hepatocellular Carcinoma ◽  
Survival Rate ◽  
P53 Protein ◽  
P53 Gene ◽  
Expression Level ◽  
Simultaneous Increase ◽  
Malignant Neoplasms ◽  
P53 Expression ◽  
Liver Cancers ◽  
Hcc Cells

Relevance: Hepatocellular carcinoma (HCC) is one of the world’s most common liver cancers and fatal malignant neoplasms. According to GLOBOCAN 2018, liver cancer ranks sixth in incidence (842,000 new cases) and fourth in mortality (782,000 deaths) globally. In Kazakhstan, there is an increase in HCC incidence from 879 cases in 2016 up to 984 cases in 2017, which amounted to 5.5 cases per 100 000 population. In 2017, the five-year survival rate was 23.7%. The mutations in the p53 gene that encodes the p53 protein are most frequent in HCC (35.2%). A high level of nuclear expression of p53 and a decrease in apoptosis of tumor cells with a simultaneous increase in their proliferative activity, noted in HCC cells, are important for the prognosis of the disease. There is a correlation between poor prognosis, a decreased survival of patients with HCC, and high expression of the p53 oncoprotein in HCC. U. Nzeako et al. have revealed a strong correlation of HCC histological differentiation with the patient survival rate. Therefore, a study of the correlation between p53 expression and HCC differentiation grade is very relevant. The purpose of this study was to analyze the correlation between p53 expression and HCC differentiation grade. Results: p53 was overexpressed in 18% of HCC cases; the expression was high in 62% and low in 20% of HCC cases. In well-differentiated HCC (G1), the p53-immunopositive nuclei expression was low in 10 cases and high in 2 cases. In moderately differentiated HCC (G2), p53 was highly expressed in 21 cases and overexpressed in 2 cases. In poorly-differentiated HCC (G3), p53 was overexpressed in 7 cases and highly expressed in 8 cases of HCC. The analysis of the correlation between p53 expression and HCC differentiation grade by Spearman showed a high correlation (r=0.79, p<0.01), evidencing a direct dependence of p53 expression level on HCC gradation. Conclusion: HCC differentiation grade highly correlates with p53 expression level in immunopositive nuclei of HCC cells. The obtained data evidences a dependence between p53 expression level and HCC gradation.

scholarly journals Inactivation of the cellular p53 gene is a common feature of Friend virus-induced erythroleukemia: relationship of inactivation to dominant transforming alleles.

1990 ◽  
Vol 10 (7) ◽  
pp. 3307-3313 ◽  
Author(s):  
D G Munroe ◽  
J W Peacock ◽  
S Benchimol
Keyword(s):  
P53 Protein ◽  
P53 Gene ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Friend Virus ◽  
P53 Expression ◽  
Virus Complex ◽  
Relationship Of ◽  
Conserved Amino Acids ◽  
Negative Phenotype

The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.

216 COMPLETE REDUCTION OF p53 EXPRESSION BY RNA INTERFERENCE FOLLOWING HETEROZYGOUS KNOCKOUT IN PORCINE FIBROBLASTS

2014 ◽  
Vol 26 (1) ◽  
pp. 222
Author(s):  
Y. J. Kim ◽  
M. J. Kim ◽  
T. H. Kim ◽  
H. W. Kim ◽  
H. Shim
Keyword(s):  
Rna Interference ◽  
Gene Targeting ◽  
P53 Protein ◽  
P53 Gene ◽  
Miniature Pig ◽  
Exon 2 ◽  
P53 Expression ◽  
Complete Reduction ◽  
Expression Levels ◽  
Fetal Fibroblasts

Because p53 has a critical role in regulation of cell cycle and apoptosis in mammals, mutations of p53 often cause various cancers in mammals. Murine models have contributed to our understanding in cancer related to p53 mutations. Mice, however, have different characteristics from humans in many ways. For instance, the short lifespan of the mouse gives rise to limitations in clinical application of the data obtained from this species. Hence, it would be beneficial to establish a more suitable model in species other than the mouse. The porcine model could be an appropriate alternative because pigs share many anatomical and physiological similarities with humans. However, the production of pigs with homozygous knockout (KO) requires years of breeding heterozygous KO animals. Here, we completely reduced the expression level of p53 mRNA and protein in miniature pig fetal fibroblasts using a combination of gene targeting and RNA interference technique. These cells may be used for nuclear transfer to directly produce pigs without expression of the gene of interest. First, we disrupted the exon 2 region of p53 gene to produce p53 heterozygous KO cells. Miniature pig fetal fibroblasts were transfected with the p53 gene targeting vector. After Geneticin treatment for 2 weeks, a total of 48 surviving colonies were screened by PCR, and one was identified as a homologous recombinant (1/48, 2.08%). Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. Transfected fibroblasts were treated with Zeocin for 3 weeks, and the shRNA integrations were confirmed by PCR. We obtained p53 KO, KD, and KOKD fibroblasts which involve p53 KO and KD either separately or simultaneously. In mRNA expression based on RT-PCR, p53 KO fibroblasts showed no difference with wild-type control (91.8 v. 100%). However, the expression levels of KD and KOKD cells significantly decreased (35.5 and 34.7%) compared with the control. In p53 protein levels analysed by Western blot, reduction of the protein was observed in p53 KD, whereas no reduction in p53 KO that might be due to heterozygous mutation. Interestingly, no p53 protein was detected in KOKD, suggesting complete reduction of the protein by synergistic effect of KO and KD. In this study, we demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference technique. Moreover, complete abolishment of protein expression is feasible using combination of the 2 techniques. In further research, cloned miniature pigs with various levels of p53 expression or even lacking p53 expression may be produced using the fibroblasts isolated in the present study as nuclear donors. These pigs may provide new animal models and insights in cancer with respect to the effect of p53.

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