Epigenetic alteration of the metallothionein 1E gene in human endometrial carcinomas.

Aberrant expression of metallothioneins (MTs) has been observed in several human tumors. In our microarray analysis, MT-1E was found to have much lower expression in endometrial cancer cells as compared with other types of cancer cells generated from the cervix, ovary or prostate. The result was confirmed by quantitative RT-PCR analysis of the MT-1E levels in individual cancer cells. Treatment of endometrial cancer cells with 5-azacytidine could reactivate MT-1E expression. We further analyzed the DNA methylation status of the promoter region of MT-1E using methylation-sensitive restriction enzymes HhaI and HpaII, followed by PCR. Promoter hypermethylation was detected in 42.4% (53/125) of the endometrial carcinoma samples, whilst none of the 38 normal tissues or hyperplasia samples were methylated. The mRNA levels of MT-1E were significantly lower in the methylation-positive than in the methylation-negative samples. Endometrial carcinoma samples with low MT-1E expression coincidently had low levels of estrogen receptor-alpha expression and vice versa. This phenomenon was not observed in the expression pattern between estrogen receptor-beta and MT-1E. There was no significant correlation between MT-1E methylation and any clinical parameters. In conclusion, a high frequency of cancer-specific hypermethylation of MT-1E was found in endometrial carcinomas. Its functional consequence in the development of endometrial cancer warrants further investigation.

P63 expression in gestational trophoblastic disease: correlation with proliferation and apoptotic dynamics.

p63 regulates cell growth and differentiation and contributes to tumorigenesis through its complex isoforms. Gestational trophoblastic disease encompasses a heterogeneous family of lesions with different malignant potential that arise from various trophoblast subpopulations. This study investigated the expression of p63 isoforms in various trophoblastic diseases and correlated with clinical progress, proliferation, and apoptotic activities. 4A4 and anti-p40 antibodies were applied to assess expressions of total and DeltaNp63 isoforms in 20 placentas, 62 hydatidiform moles, 9 choriocarcinomas, 5 placenta site trophoblastic tumors, and 2 epithelioid trophoblastic tumors immunohistochemically. The immunoreactivity of p63 was localized to the nuclei of cytotrophoblast, villous, and chorionic-type intermediate trophoblasts with significant correlation between 2 p63 indices (P<0.001). p63 indices were significantly lower in placentas of advanced gestational age (P<0.001). Hydatidiform moles demonstrated significantly higher p63 indices than normal placentas (P<0.001). Epithelioid trophoblastic tumors displayed the highest p63 indices (45%-80%) whereas immunoreactivity was only focal in choriocarcinoma (0%-5.62%) and was essentially absent in placenta site trophoblastic tumors. There was no significant correlation between p63 indices and subsequent development of trophoblastic neoplasia in hydatidiform moles (P>0.05). Both p63 indices positively correlated with the proliferative index (Ki67) (P<0.05), apoptotic index (M30) (P<0.005), p53 (P<0.005), and p21(WAF1/CIP1) expression (P<0.005). Our results indicate that DeltaNp63, the dominant isoforms expressed in trophoblasts, display heterogeneous expression patterns in relation to trophoblast subtypes. We also demonstrate for the first time the possible role of p63 in the pathogenesis of gestational trophoblastic disease (GTD) through its interaction with p53-dependent proliferation and apoptotic activities.

Expression of deltaNp73 and TAp73alpha independently associated with radiosensitivities and prognoses in cervical squamous cell carcinoma.

PURPOSE: The p73 gene produces different protein isoforms using alternative promoters and splicing, which have different biological characteristics. This study was to investigate the expression patterns of two distinct p73 isoforms (deltaNp73 and TAp73alpha) in cervical squamous cell carcinomas (SCC) and the relationship between their expressions and prognostic significance in cervical SCC patients. EXPERIMENTAL DESIGN: We investigated the protein expressions of deltaNp73 and TAp73alpha in 117 cervical SCC and 113 normal cervical tissues using immunohistochemistry. The expression levels were analyzed with clinical variables and patients' survival. RESULTS: DeltaNp73and TAp73alpha were significantly overexpressed in cervical SCC compared with those in normal cervical epithelium (P < 0.001). However, their expressions were inversely correlated (P < 0.001, R = -0.368) and associated with differential tumor radiosensitivity. Overexpression of deltaNp73 was significantly found in SCC resistant to irradiation (P < 0.001), whereas increase of TAp73alpha expression was observed in the majority of SCC sensitive to irradiation (P < 0.001). Multivariate and survival analyses indicated that the expressions of deltaNp73 and TAp73alpha were independently associated with prognosis: deltaNp73 was associated with recurrence of the disease [P = 0.001; odds ratio (OR), 4.857] and an adverse outcome (P = 0.012; OR, 4.676), whereas TAp73alpha predicted a better survival of cervical SCC patients (P = 0.018; OR, 0.065). CONCLUSIONS: The p73 gene might be an important determinant of cellular response to irradiation. The expressions of the two main isoforms (deltaNp73 and TAp73alpha) might be potential markers for predicting the prognosis and sensitivity to radiotherapy in patients with cervical SCC.

Label-free colorimetric detection of cancer related gene based on two-step amplification of molecular machine.

Highly sensitive detection of K-ras gene is of great significance in biomedical research and clinical diagnosis. Here, we developed a colorimetric biosensing system for the detection of proto-oncogene K-ras based on enhanced amplification effect of DNA molecular machine, where dual isothermal circular strand-displacement amplification (D-SDA) occurs on two arms in one-to-one correspondence. Specifically, we designed a primer-locked hairpin probe (HP) and a primer-contained linear polymerization template (PPT). In the presence of target gene, HP can hybridize with PPT, forming a DNA molecular machine with dual functional arms (called DFA-machine). Each of the two probes in this machine is able to be extended by polymerase on its counterpart species. Moreover, with the help of nicking endonuclease, the dual isothermal polymerization is converted into dual circular strand-displacement amplification, generating a large amount of anti-hemin aptamer-contained products. After binding to hemins, the aptamer/hemin duplex, horseradish peroxidase (HRP)-mimicking DNAzyme, was formed and catalyzed the oxidation of colorless ABTS by H2O2, producing a visible green color. The proposed colorimetric assay exhibits a wide linear range from 0.01 to 150nM with a low detection limit of 10pM. More interestingly, the mutations existing in target gene are easily observed by the naked eye. It should be noted that this colorimetric system was proved by the analysis of K-ras gene of SW620 cell lines. The simple and powerful DFA-machine is expected to provide promising potential in the sensitive detection of biomarkers for cancer diagnosis, prognosis and therapy.

[Clinicopathological study of intermediate trophoblastic non-tumor lesions: exaggerated placental site and placental site nodule].

OBJECTIVE: To investigate the clinicopathological features of intermediate trophoblastic non-tumor lesions, and to evaluate the position of immunohistochemistry in differential diagnoses. METHODS: Clinical presentation and morphological study of 15 cases of exaggerated placental site (EPS) and 4 cases of placental site nodule or plaque (PSNP) were reviewed. Immunohistochemical stains for hCG, hPL, inhibin-alpha, PLAP, CK18 and Ki-67 were performed. RESULTS: The age of patients ranged from 25 to 40 years with an average of 31.5 years for EPS and 26 to 39 years with an average of 34.3 years for PSNP. Microscopically, EPS was characterized by cords and small sheets of implantation site intermediate trophoblasts infiltrating the endometrium, myometrium and arterial walls. The general histological structures of the endometrium and myometrium were preserved. PSNP was characterized by multiple circumscribed nodular lesions consisting of so-called chorionic-type intermediate trophoblasts and hyaline-like matrix present in the endometrium. Immunohistochemical stainings for hPL and CK18 were positive in the 15 EPS cases. Immunoreactivity for CK18, Inhibin-alpha and PLAP was detected in 4 PSNP cases. The Ki-67 labeling index in 15 EPS cases was low (< or = 5%), while Ki-67 index in 4 PSNP cases was close to 0. CONCLUSIONS: The clinical presentation and pathological features of EPS and PSNP differ from those of trophoblastic tumors (placental site trophoblastic tumor, epithelioid trophoblastic tumor and choriocarcinoma). Immunochemical staining is of great value in their differential diagnoses.