Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene.

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC-MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.

Inverted mirror image molecular beacon-based three concatenated logic gates to detect p53 tumor suppressor gene.

Mutation of p53 tumor suppressor gene represents one of the early molecular events in tumor initiation and progression. Although molecular computing holds tremendous potential with important applications in diagnosis, prognosis and treatment of human diseases at the molecular level, designing molecular logic gates to implement cascade amplification via operating autonomously for the detection of point mutations still remains challenging. In this contribution, we developed a three concatenated logic gates (TCLG) to perform multiple strand displacement amplification (m-SDA) for screening the cancer-related point mutations only via designing an innovative molecular beacon (MB). Specifically, using p53 gene as model target, extending the two ends of a MB via adding two fragments with the same sequence achieves two unique terminal single-stranded (ss) overhangs. After self-folding of MB into hairpin structure, the two overhangs exhibit a near inverted mirror image (IM) relationship if taking the base nature and direction into account. For this, the probe is called IM-MB. Because cascade SDAs can occur on IM-MB and promote each other, the target gene can be detected down to 10 pM. Along this line, the TCLG circuit was proposed, and two primers and target gene serve as the indispensable input signals. Utilizing this logic circuit, the point mutation or absence of target gene can be sensitively screened. Moreover, its potential application in the recognition of point mutations in complex biomatrix has been demonstrated via blind test. The proof-of-concept scheme is expected to provide new insight into the development of DNA-based molecular logic gates and their applications in basic research, medical diagnosis and precise treatment and treatment of genetic diseases.

Data on the putative role of p53 in breast cancer cell adhesion: Technical information for adhesion assay.

In this data article, the potential role of p53 tumor suppressor gene (p53) on the attachment ability of MCF-7 breast cancer cells was investigated. In our main article, "IGF-I/ EGF and E2 signaling crosstalk through IGF-IR conduit point affect breast cancer cell adhesion" (K. Voudouri, D. Nikitovic, A. Berdiaki, D. Kletsas, N.K. Karamanos, G.N. Tzanakakis, 2016) [1], we describe the key role of IGF-IR in breast cancer cell adhesion onto fibronectin (FN). p53 tumor suppressor gene is a principal regulator of cancer cell proliferation. Various data have demonstrated an association between p53 and IGF-IR actions on cell growth through its' putative regulation of IGF-IR expression. According to our performed experiments, p53 does not modify IGF-IR expression and does not affect basal MCF-7 cells adhesion onto FN. Moreover, technical details about the performance of adhesion assay onto the FN substrate were provided.

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus.

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1-3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.

No association of p53 codon 72 polymorphism with idiopathic recurrent pregnancy loss in Korean population.

OBJECTIVE: The balance of apoptosis and proliferation is an important part in the embryonic development during pregnancy. It has been reported that the p53 gene plays a significant role in angiogenesis and placental development, namely in reproduction and is suggested as a potential mediator of pregnancy. This study was performed to investigate whether the genetic polymorphism of the p53 gene is associated with idiopathic recurrent pregnancy loss (RPL). STUDY DESIGN: We conducted a case-control study in the Korean population. Study subjects consisted of 294 patients with idiopathic RPL and 300 postmenopausal controls. The genotyping for the p53 codon 72 polymorphism was performed using a Taqman assay. Continuous variables were compared using Student's t test and the χ(2) test was used to evaluate differences in the genotype distributions between the RPL and the controls. RESULTS: There were no significant differences in the genotype distributions or allele frequencies of the p53 codon 72 polymorphism between the RPL and control group. There was also no significant association between the p53 codon 72 polymorphism and RPL risk in both recessive (Pro/Pro vs. Arg-carriers, p=0.314) and dominant model (Pro-carriers vs. Arg/Arg, p=0.383: data not shown). CONCLUSION: The codon 72 polymorphism in the p53 gene did not show any correlation with idiopathic RPL in Korean women, implying that it may not be susceptible allelic variants or be insufficient to cause RPL.

Hipermetilación del gen supresor de tumores p53 en pacientes pediátricos con leucemia linfoblástica aguda / Hypermethylation of tumor suppressor gene p53 in Chilean pediatrics patients under 15 years with acute lymphoblastic leukemia

La leucemia linfoblástica aguda (LLA) es la neoplasia maligna hematooncólogica más frecuente en pacientes pediátricos contando hasta 75% de las leucemias y 32-35% del total de cánceres infantiles. Aunque la LLA es considerada una enfermedad con base genética, es cada vez más evidente que alteraciones epigenéticas desempeñan un rol central en su patogénia y progresión. La hipermetilación de regiones promotoras de genes es asociada con la pérdida de función génica. El gen supresor de tumores p53 (GST), es uno de los principales genes en el ciclo celular y apoptosis. El objetivo de este trabajo fue determinar el estado de metilación en la región del promotor-exón 1 del GST p53 y la asociación con la supervivencia en menores de 15 años con LLA. Se analizaron 40 pacientes provenientes de la Región de la Araucanía-Chile. La hipermetilación del p53 se determinó combinando enzimas de restricción sensibles a metilación (HpaII y EcoR II) y reacción en cadena de la polimerasa. Los resultados indicaron que 15/40 casos (37,5%) presentaron hipermetilación. Se encontró una diferencia estadística en la supervivencia según estado de metilación de p53 en el grupo de niñas (p=0,02). Considerando el total de pacientes, una tendencia a mejor supervivencia cuando los recuentos de leucocitos fueron <30.000/mm3 (p=0,08). Se encontró frecuentemente hipermetilado el gen p53 en la región del promotor-exon1. Esto indicaría que la hipermetilación del GST p53 puede ser un evento importante en la patogénesis de la LLA.

Acute lymphoblastic leukemia (ALL) is the most common hematology oncology malignancy in pediatric patients counting up to 75% of leukemias and 32­35% of all childhood cancers. Although ALL is considered a disease with a genetic basis, it is increasingly clear that epigenetic alterations play a central role in the pathogenesis and work was to determine the methylation status in promoter-exon1 of the TSG-p53 and association with survival in children under 15 years with ALL. In our study 40 patients from the Araucanía Region, Chile were analyzed. Hypermethylation of p53 was determined by combining restriction enzymes sensitive to methylation (HpaII and EcoR II) and polymerase chain reaction. Results indicated that 15/40 cases (37.5%) showed hypermethylation. Statistical difference was found in survival according to p53 methylation status in the girls group (p=0.02). Considering all patients, there was a trend to improved survival when leukocyte counts were <30.000/ul (p=0.08). We found the p53 gene frequently hypermethylated in the promoter-exon1 region. This would indicate that TSG p53 hypermethylation may be an important event in the pathogenesis of ALL.

Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

The Individual Cell Properties of Oral Squamous Cell Carcinoma and p53 Tumor Suppressor Gene Mutation / Oral Science International

There is no consensus on the relationship between variations in TP53 mutations and tumor properties in oral squamous cell carcinoma (OSCC). To further the basic research required to eventually develop individualized (order-made) treatments and prognoses for OSCC, we established six human OSCC lines from patients within our department. Together with another nine cell lines derived from donations by other organizations, we determined the TP53 mutation and single nucleotide polymorphism (SNP) of codon 72 in a total of 15 cell lines, and examined <i>in vitro</i> cell invasion activity and anti-cancer drug sensitivity. The missense mutation at codon 248 was most abundant, and was noted in four cell lines, but other diverse mutation variations were also revealed. The cells which expressed the mutated p53 protein (the p53 (+) group) showed slightly higher invasion activity than did the p53 (-) group. In p53 (+) group, the 72R of SNP (72P/R) was higher than the 72P in invasion activity, although the difference was not significant. Surprisingly, an anti-cancer drug sensitivity test with four different types of drugs showed that the p53 (-) group was more resistant in other than CDDP, and that 72R was more sensitive than 72P in the p53 (+) group. To clarify the characteristics of the R248Q mutation, which is the most abundant missense mutation, the gene was introduced with an expression plasmid vector into a TP53 null Saos-2 cell. The transformant of R248Q mutation gained higher activity of invasion, while its anti-cancer drug sensitivity also increased. Our findings suggest that it may be possible to estimate oral cancer cell characteristics and the malignancy level based on differences in the TP53 mutation.

Combination treatment for osteosarcoma with baculoviral vector mediated gene therapy (p53) and chemotherapy (adriamycin)

The insect baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been evaluated as a vector for gene delivery to human tumor cells. A human osteogenic sarcoma cell line, Saos-2, was found to be highly susceptible to infection with a baculoviral vector, with nearly 100% of Saos-2 cells being able to express a lacZ reporter gene after a brief exposure to the virus at a m.o.i. of 30 pfu/cell. The production of beta-galactosidase protein was 18-times greater than that in HepG2 cells which were previously thought to be the mammalian cells most susceptible to the baculovirus. The possibility of developing a baculovirus as a cytotoxic vector for p53-defective cancer was tested by destruction of Saos-2 cells (p53-/-) with a recombinant baculovirus containing the wild type p53 gene (BV-p53) in vitro. The p53 baculovirus induced apoptotic cell death in tumor cells in a dose-dependent manner with approximately 60% killing at an m.o.i. of 160 pfu/cell. Combined treatments of gene therapy (p53) and chemotherapy (adriamycin) resulted in synergistic and potent killing of the osteogenic sarcoma cells. For example, greater than 95% of Saos-2 cells were killed by the combination of BV-p53 (m.o.i. of 100) and adriamycin (35 ng/ml), whereas approximately 50% and approximately 55% cells were killed by BV-p53 and adriamycin alone, respectively. These results indicate that a baculoviral gene delivery vector can be used to efficiently target certain types of mammalian cells and the combination treatment of gene-therapy mediated by a baculovirus and chemotherapy may enhance induction of apoptosis in cancer cells.

Effects of Wild - type p53 Gene Transfection into Human Colon Cancer Cell Line / 대한암학회지

PURPOSE: In colon cancer, the most frequent genetic alteration is found in p53 tumor suppressor gene residing on the short arm of chromosome 17. In order to investigate the significance of wild-type p53, we transfected wild type p53 into human colon cancer cell lines and analysed tbeir biologic effects. MATERIALS AND METHODS: For analysis of p53 status in cell lines, polymerase chain reaction-single stranded confonnation polymorphism (PCR-SSCP), PCR-direct sequencing and Western blot analysis were employed. Transient transfection with liposome-p53 complex was followed by cell biologic assay. RESULTS: We found that twelve of fifteen human colon cancer cell lines showed mutation of p53 by PCR-SSCP method. These results almost corresponded to p53 protein accumulations assessed by Westem blot using PAbl801. After transfection with lipafect- AMINE and wild type p53 complex on p53 mutant type cell line (LS1034), viability was reduced to 17.9%, and invasiveness was reduced to 37.3%. Morphologically, wild type p53 transfected cells showed lumen formation and apoptosis after induction of differentiation by Matrigel. CONCLUSION: Wild type p53 transfection into p53 mutated colon cancer ceil line resulted in restoration of tumor suppressor effect of p53, and this model would be one of the experimental systems for p53-based gene therapy.

The effects of wild type p53 tumor suppressor gene expression on the normal human cervical epithelial cells or human epidermal keratinocytes transformed with human papillomavirus type 16 DNA

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.

Role of human papillomavirus and p53 tumor suppressor gene in cervical carcinogenesis

To determine whether the dysfunction of p53 protein, caused either by the mutation of p53 gene itself or by the human papillomavirus (HPVs) is involved in the development of cervical cancers and to find out the status of p53 tumor suppressor gene in HPV positive or negative cancers, we analyzed 64 cases of primary cervix cancers. First, polymerase chain reaction (PCR) was performed with E6 consensus primer pairs to detect the infection of HPVs in cervix cancer tissues. Second, to screen the p53 gene mutation, PCR of p53 exon 5 through 9 and single strand conformation polymorphism (SSCP) analysis were performed with p53 exon specific primers. Finally, overexpression of p53 protein was checked by immunohistochemical staining with anti p53 antibody. We found HPVs in 43 cases out of 64 cases (67.2%). HPV type 16 was positive in 26 cases, type 18 was positive in 7 cases, and 6 cases were positive for both HPV type 16 and 18. HPV type 31 or 33 was not detected in our experiments, and in 4 cases, type of HPVs were unknown. The SSCP analysis showed mobility shifts in 8 cases (6 in HPV positive cases; 2 in HPV negative cases) and the sequence analysis confirmed the results of SSCP analysis. In addition, the overexpression suggesting the mutation of p53 gene was detected in 27 cases (42.2%, 21 in HPV positive cases; 6 cases in HPV negative cases). Our results support the previous reports that the dysfunction of p53 plays an important role in the development of cervix cancers but contrary to the results obtained from cervix cancer all lines, there is no inverse correlation between HPV infections and p53 mutations in primary cervix cancers.

Analysis of p53 tumor suppressor gene mutations and human papillomavirus infection in human bladder cancers

To determine whether the dysfunction of p53 caused either by mutation of the p53 gene itself or by binding to E6 protein of oncogenic HPVs is involved in the transitional cell carcinomas (TCCs) of the bladder, we analyzed 23 TCCs of the bladder. DNA was extracted from each paraffin embedded tissue of TCCs of bladder and polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) analysis were performed to screen mutations in p53 tumor suppressor gene, then PCR/dot blot hybridization were performed to detect infection of HPVs. We found that p53 gene mutation was found in 3 cases and oncogenic HPV infection was detected in 8 cases and thus, the overall incidence of possible p53 dysfunction was 47.8% on DNA analysis (If the results of immunohistochemistry to detect overexpression of p53 protein were included, the incidence was 60.9%). Therefore, we concluded that dysfunction of p53 plays a major role in the development of TCCs of bladder in Korean patients.