Overexpression of prostate stem cell antigen is associated with gestational trophoblastic neoplasia.

AIMS: Hydatidiform mole (HM) is the most common type of gestational trophoblastic disease. A proportion of patients with HM develop gestational trophoblastic neoplasia (GTN) requiring chemotherapy. The aim was to identify differentially expressed genes that are associated with development of GTN. METHODS AND RESULTS: Using cDNA microarray, differential expression of prostate stem cell antigen (PSCA) was identified in HMs that developed GTN compared with those that spontaneously regressed. Significant overexpression of PSCA RNA (P = 0.037) and protein (P < 0.05) in aggressive HM was verified by real-time polymerase chain reaction (PCR) and immunohistochemical analysis in 10 first-trimester placentas, 36 HM that subsequently regressed and 11 HM that developed GTN. A high level of PSCA expression was also found in three choriocarcinomas and three placental site trophoblastic tumours. A positive correlation was observed between PSCA expression and proliferation and apoptotic indices as assessed by Ki67 (P = 0.01), mcm7 (P = 0.001) and M30 (P = 0.016), as well as p53 (P < 0.01), p21(WAF1/CIP1) (P < 0.01) and mdm2 (P = 0.002) expression. CONCLUSIONS: Overexpression of PSCA is associated with development of GTN in HM. PSCA probably plays a role in the regulation of cell growth through p53-related signaling pathways.

[Phenotypic and genotypic features of twenty children with classic pantothenate kinase-associated neurodegeneration].

Objective: To explore the phenotypic and genotypic characteristics in Chinese children with classic pantothenate kinase-associated neurodegeneration (PKAN). Method: The clinical, radiographic and genetic data of all PKAN patients diagnosed at pediatric department of Peking University First Hospital from November 2006 to December 2016 were retrospectively collected and analyzed. Result: Twenty patients with classic PKAN were included in the study. The median age at onset was 3.5 years (ranging from 1.0 to 10.0 years), and the most common initial symptom was gait disturbance (16 cases). At the last evaluation, the clinical features were limbs dystonia (20 cases), dysarthria (16 cases), dysphagia (11 cases), pyramidal sign (7 cases), mental regression (3 cases) and pigmentary retinopathy (5 cases). For those classic PKAN patients, the median time from onset of disease to loss of independent ambulation was 6.9 years (ranging from 2.0 to 12.0 years). Imaging data showed, except "eye of tiger" in MRI (19 cases), globus pallidus calcification in CT was also found in four patients. In gene testing, 26 different mutations in PANK2 gene were identified, and 16 of 26 were novel mutations. Moreover, c. 1502T>C (p.Ile501Asn) was the most common mutation (4 cases). Conclusion: Dystonia is the major neurologic feature of classic PKAN. Disease progression is rapid, with loss of independent ambulation within 10 years after onset. Except "eye of tiger" in MRI, globus pallidus calcification in CT may be another imaging feature of PKAN.Sixteen novel mutations of PANK2 gene were identified in the study.

Differential gene expression identified in complete hydatidiform mole by combining suppression subtractive hybridization and cDNA microarray.

Complete hydatidiform mole (CHM) is a type of gestational trophoblastic disease with pure paternal chromosome contribution and unpredictable malignant potential. As an attempt to assess the molecular pathogenesis of CHM, suppression subtractive hybridization (SSH) combined with cDNA microarray was used to compare the gene expression pattern of CHM compared with normal first-trimester placenta of similar gestational ages. cDNA microarray analysis using tissue-specific chips constructed with subtracted cDNA libraries identified 13 differentially expressed gene transcripts. Quantitative real-time polymerase chain reaction (PCR) confirmed up-regulation of human chorionic gonadotropin beta subunit (CGB) (P=0.0008) and KIAA1200 (P=0.0005), a G-protein regulator, as well as down-regulation of osteopontin (SPP1) (P<0.0001) in 14 genotyped CHM when compared with 15 normal placentas. These candidate genes may contribute toward understanding the mechanism involved with the development and progression of CHM.

Effect of all-trans retinoic acid on tissue dynamics of choriocarcinoma cell lines: an organotypic model.

BACKGROUND: All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death. AIM: To investigate the tissue dynamic and cellular invasion effects of ATRA in choriocarcinoma (CCA), an aggressive trophoblastic tumour, by using a three-dimensional organotypic culture model system and cell invasion assay, respectively. METHODS: An organotypic culture model of two CCA cell lines, JAR and JEG, was established. The effects of 1 microM ATRA on proliferation, differentiation and apoptosis on this CCA model were assessed by morphological assessment of the mitotic and apoptotic figures as well as by Ki-67 and caspase-related M30 cytoDeath antibody immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The effect of ATRA on p53 and its regulated protein product, WAF1/Cip1, was also evaluated with DO7 and p21(WAF1) antibodies, respectively. Moreover, the effect of ATRA on cellular (CCA) invasion was also investigated with Cell Invasion Kit on the JEG cell line. RESULTS: ATRA was found to induce marked apoptosis in organotypic cultures of both cell lines, as evidenced by increased M30-positive cells (p<0.0001) and increased TUNEL-positive cells (p<0.0001) in treated cultures; to decrease proliferation, as evidenced by decreased Ki-67-positive cells (p<0.0001); and to decrease p53-DO7 immunoreactivity (p<0.0001) and increase p21(WAF1) (p<0.0001) immunoreactivity. 1.5 microM ATRA was found to effectively inhibit JEG cell invasion in the cell invasion assay. CONCLUSION: ATRA treatment was found to inhibit invasion and proliferation and enhance apoptosis, probably by the activation of caspases and induction of differentiation. ATRA and synthetic retinoids may be alternative agents for the treatment of CCA.

Epstein-Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells.

Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase.

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

Id helix-loop-helix proteins are differentially expressed in gestational trophoblastic disease.

AIMS: To assess the expression of Id proteins in trophoblastic tissues and to correlate this with clinical parameters, proliferative and apoptotic indices as well as to related oncogene expression. METHODS AND RESULTS: Immunohistochemistry for Id1, Id2, Id3 and Id4 was performed on 83 trophoblastic tissues including 17 normal first-trimester placentas, seven term placentas, 47 hydatidiform moles (HM), and 12 spontaneous miscarriages. The four Id proteins were predominantly expressed in the villous and implantation site intermediate trophoblast. Expression of Id1 in HM was significantly higher than that in normal placenta (P = 0.0006) and spontaneous miscarriage (P = 0.0001) but did not correlate with subsequent development of gestational trophoblastic neoplasia (GTN). Id1 expression correlated with the proliferation index as assessed by MCM7 (P = 0.003) and Ki67 (P = 0.017) and with the apoptotic activity assessed by TUNEL (P = 0.001) and M30 CytoDeath antibody (P = 0.013). Moreover, the expression of Id1 correlated with the expression of p53 (P = 0.004), p21(WAF1) (/CIP1) (P = 0.003) but not with p16 (P = 0.107). CONCLUSIONS: Id proteins may play a role in the regulation of proliferative and apoptotic activity in trophoblastic tissue and are potentially useful in differentiating molar and non-molar gestation, but are not helpful in predicting GTN.

Regulation of oxytocin receptor gene expression in sheep: tissue specificity, multiple transcripts and mRNA editing.

The increase in uterine oxytocin receptor concentrations over the late luteal phase of the oestrous cycle in sheep is thought to play an important role in the regulation of the duration of the cycle by facilitating the effect of oxytocin on uterine prostaglandin release. Experiments indicated that oxytocin receptor mRNA expression in the endometrium was high at oestrus compared with at days 2, 7 and 12 of the oestrous cycle. The amount of oxytocin receptor mRNA expression in the pituitary gland did not show any significant differences during the oestrous cycle. Oxytocin receptor cDNA was obtained and characterized from ovine uterine endometrium on day 15 of the oestrous cycle, using RT-PCR techniques, to study the mechanisms underlying the resolution of oxytocin receptor expression. The cDNA sequence for the oxytocin receptor gene in sheep was found to be similar to that described previously, except for a difference of seven nucleotides. These nucleotide differences resulted in changes in four of the deduced amino acids in the oxytocin receptor sequence. The heterogeneity of the different sized oxytocin receptor transcripts in sheep is due, at least in part, to the alternative use of polyadenylation sites. Northern hybridization confirmed that the oxytocin receptor gene is expressed in ovine corpus luteum. The investigations on oxytocin receptor gene expression indicate that the patten of oxytocin receptor gene expression in sheep is not only tissue-specific, but also highly function-related. Evidence was obtained of mRNA editing in both the coding and the 3'-untranslated (3'UTR) regions of oxytocin receptor gene transcripts in ovine endometrium; this was the first demonstration of this phenomenon for oxytocin receptor mRNA. The present results indicate that the observed differences in oxytocin receptor mRNA sequences for the different oxytocin receptor populations in endometrium are due to mRNA editing. mRNA editing of oxytocin receptor transcripts may be reflected in changes in the amino acid composition of the carboxyl terminus of the receptor, which would explain the differences in the observed responses to an oxytocin challenge.

Methylation status and expression of E-cadherin and cadherin-11 in gestational trophoblastic diseases.

The clinical significance of cadherins in gestational trophoblastic diseases (GTD) is not fully understood. In this study, the expression of E-cadherin and cadherin-11 in 12 normal placentas, 32 cases of hydatidiform mole (HM) including 15 complete HMs and 17 partial HMs, and five choriocarcinomas was investigated by immunohistochemistry and correlated with follow-up of HMs. Cases with available frozen blocks were further analyzed by western blot and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Methylation of E-cadherin was investigated by methylation-specific PCR in six normal first trimester placentas, 19 HMs and their associated deciduas. E-cadherin expression was localized to cytotrophoblast and intermediate trophoblast whereas cadherin-11 was expressed in syncytiotrophoblast, intermediate trophoblast, and decidua. Immunoreactivity of E-cadherin was reduced in choriocarcinoma and complete HM when compared with that in normal first trimester placenta (P < 0.01, P = 0.04). Hypermethylation of E-cadherin was demonstrated in three complete HMs with the lowest level of E-cadherin. Compared with normal first trimester placenta, immunoreactivity of cadherin-11 was higher in complete HM (P = 0.02), but lower in choriocarcinoma (P = 0.02). Such differential expression was confirmed by western blot and semiquantitative RT-PCR. No obvious association was observed between the development of persistent trophoblastic disease with the expression of E-cadherin and cadherin-11.