Extraskeletal osteosarcoma of the orbit: A clinicopathologic case report and review of literature.

Primary extraskeletal osteosarcoma (EOS) is an extremely rare malignancy. In this report, the clinical course of a 32-yearold man presenting with proptoses is described. Medical history included Hirschsprung disease (HD), horseshoe kidney, azoospermia, and vertebral anomalies. Imaging of the orbit showed an oval, well-defined heterogeneous mass adjacent to the lateral wall of the orbit. The patient underwent a lateral orbitotomy and complete excision of the mass. The mass was not attached to the bone. Histopathologic and immunohistochemical examination confirmed the diagnosis of an EOS. The patient received chemotherapy and radiotherapy and is free of the disease 3 years after the diagnosis. Genetic screening showed no mutations for both the RET protooncogene for HD and the p53 tumor suppressor gene for osteosarcoma.

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.

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.

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.

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.

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.