Sai-Wah Tsao

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 p16INK4A locus and downregulation of RASSF1A expression. The deletion of the p16INK4A 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 p16INK4A locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF‐κB 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.

Promoter Hypermethylation of Multiple Genes in Hydatidiform Mole and Choriocarcinoma

The methylation status of genes in hydatidiform mole and choriocarcinoma and its significance is relatively unexplored. We investigated the methylation status of the promoter regions of six genes, p16, HIC-1, TIMP3, GSTP1, death-associated protein kinase (DAPK), and E-cadherin in 54 hydatidiform moles, five choriocarcinomas, and 10 first trimester placenta by methylation-specific polymerase chain reaction (PCR). Immunohistochemical expression of p16, TIMP3, and E-cadherin, and quantitative real-time RT-PCR of p16 was also performed. Among the six genes examined, the promoter region of four genes (E-cadherin, HIC-1, p16, TIMP3) in choriocarcinoma and three genes (E-cadherin, HIC-1, p16) in hydatidiform mole exhibited aberrant methylation whereas none was hypermethylated in normal placenta. There was a significant correlation between methylation and reduced expression of p16, E-cadherin, and TIMP3 (P < 0.001). Fifteen of the 54 patients with hydatidiform mole developed gestational trophoblastic neoplasia requiring chemotherapy. Promoter hypermethylation of p16 alone, or combined with E-cadherin, was significantly correlated to such development (P = 0.001, 0.0005, respectively). Hypermethylation of multiple genes, especially p16, might be related to the subsequent development of gestational trophoblastic neoplasia.

Prevalence of HPV infection in esophageal squamous cell carcinoma in Chinese patients and its relationship to the p53 gene mutation

Human papillomavirus (HPV), in particular types 16 and 18, is positively associated with anogenital cancers and may be an important etiologic factor in their pathogenesis. The goal of our study was to investigate the role of HPV infection in the pathogenesis of esophageal squamous cell carcinoma (ESCC) and its relationship with the p53 mutation. We have examined ESCC collected from Sichuan, China, for the presence of HPV infection and p53 mutation. The presence of HPV DNA was detected by PCR‐Southern analysis while the p53 mutation was analyzed by PCR‐SSCP. High‐risk HPV (types 16 and 18) DNA was detected in 32 out of 152 cases of ESCC examined. In contrast, HPV DNA was not detected in normal esophageal tissues excised from the distant end (tumor free) of resected ESCC. Mutation of the p53 gene was detected in 22 out of 55 cases of ESCC. The distribution of the 22 p53 mutation was: 5 in exon 5, 1 in exon 6, 5 in exon 7, 10 in exon 8 and 1 in exon 10. The p53 mutation was detected at a significantly lower rate in ESCC with HPV infection. Our results support a role of HPV infection in the pathogenesis of ESCC from a high‐incidence area. Int. J. Cancer 72:959–964, 1997.

Analysis of gestational trophoblastic disease by genotyping and chromosome in situ hybridization.

Hydatidiform mole is classified into partial and complete subtypes according to histopathological and genetic criteria. Distinction between the two by histology alone may be difficult. Genetically, a complete mole is diploid without maternal contribution, whereas a partial mole is triploid with a maternal chromosome complement. To assess the accuracy of histological diagnosis by correlating with the genetic composition, we performed fluorescent microsatellite genotyping to detect the presence or absence of maternal genome in a hydatidiform mole and carried out chromosome in situ hybridization to analyze the ploidy. For genotyping analysis, paraffin sections of 36 complete and nine partial moles, diagnosed according to histological criteria, were microdissected and DNA was separately extracted from the decidua and molar villi. Six pairs of primers that flank polymorphic microsatellite repeat sequences on five different chromosomes were used. In all, 34 cases, including 31 complete moles and three partial moles diagnosed histologically, showed no maternal contribution by genotyping; thus these could be genetically considered as complete mole. The other 11 cases (five complete moles and six partial moles previously diagnosed by histology) showed the presence of maternal contribution and were genetically diagnosed as partial moles. The genotyping results correlated with histological evaluation in 88% (37/45) of hydatidiform mole and correlated with chromosome in situ hybridization findings in all the cases, that is, triploid hydatidiform moles had maternal-derived alleles, while diploid hydatidiform moles were purely androgenetic. Compared with genetic diagnosis, histological evaluation was more reliable for the diagnosis of a complete mole (91%, 31/34) than that of a partial mole (55%, 6/11) (P=0.0033). Seven complete moles and three partial moles diagnosed genetically developed gestational trophoblastic neoplasia. To conclude, genotyping and chromosome in situ hybridization can provide reliable adjunct to histology for the classification of a hydatidiform mole, especially in cases with difficult histological evaluation and early gestational age. As a partial mole still carries a risk of developing gestational trophoblastic neoplasia, follow-up is considered necessary for both complete and partial moles.

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis.

Metastatic trophoblastic disease after an initial diagnosis of partial hydatidiform mole: genotyping and chromosome in situ hybridization analysis.

Hydatidiform mole (HM) is classified into partial (PHM) and complete (CHM) subtypes according to histopathologic and genetic criteria. Traditionally, it is believed that PHM carries a better prognosis and rarely develops metastasis. However, making a distinction between PHM and CHM using histologic criteria alone may be difficult.

Correlation of increased apoptosis and proliferation with development of prostatic intraepithelial neoplasia (PIN) in ventral prostate of the Noble rat

Imbalance between cell proliferation and cell apoptosis has been considered a key factor in carcinogenesis. Prostatic intraepithelial neoplasia (PIN) is the most likely precancereous lesion and represents the major target for chemoprevention of prostate cancer. The proliferative and apoptotic activities involved in the development of PIN remain to be elucidated.

Minichromosome maintenance protein 7 expression in gestational trophoblastic disease: correlation with Ki67, PCNA and clinicopathological parameters

Aims:u2002 To assess the proliferative activity of gestational trophoblastic disease (GTD) using one of the novel proliferation markers (MCM7) and to determine its prognostic value in hydatidiform mole (HM).

Effect of insulin-like growth factor 1 on PHA-stimulated cord blood mononuclear cell telomerase activity

Telomerase may contribute to the capacity for cell replication by compensating for the loss of telomere length. Exploring the use of biological modifiers in increasing cellular replicative potential through telomerase activity may be useful for in vitro expansion of haemopoietic stem cells for transplantation or lymphoid cells for adoptive immunotherapy. In this study we showed for the first time that insulin‐like growth factor 1 (IGF‐1) modulated telomerase activity in human cord blood mononuclear cells (MNC) and some of the known functional determinants of telomerase activity. We found that cord blood MNC expressed constitutively a low level of telomerase activity and human telomerase reverse transcriptase (hTRT) mRNA, and a high level of human telomerase RNA component (hTR) and telomerase‐associated protein‐1 (TP1) mRNA. Interestingly, IGF‐I alone did not increase the telomerase activity of cord blood MNC but could enhance the PHA‐induced increase in telomerase activity. These alterations in telomerase activity were not completely in phase with those of proliferation response. On the other hand, IGF‐I did not alter hTRT mRNA expression but enhanced the PHA‐induced increase in hTRT whereas TP1 mRNA expression was stimulated by either IGF‐I or PHA but showed no additive increase when stimulated by both IGF‐1 and PHA. Neither IGF‐1 nor PHA altered hTR expression. Finally, the temporal dynamics of hTRT mRNA expression and telomerase activity in cord blood MNC over 5 d in culture were not totally concordant, suggesting that key factors other than hTRT were involved in regulating telomerase activity in cord blood MNC. The modulatory effect of IGF‐1 on telomerase activity supports its potential role in increasing replicative potential of cord blood lymphoid cells or haemopoietic stem cells.

Presence of human papillomavirus in esophageal squamous cell carcinomas of Hong Kong Chinese and its relationship with p53 gene mutation.

There is no scientific study that has investigated the association between human papilloma virus (HPV) and p53 mutation in Hong Kong Chinese patients with esophageal cancers. The aim of this survey is to evaluate in details the prevalence and relationship of HPV and p53 mutation in these patients with esophageal squamous cell carcinomas. Fresh tissues from the resected specimens of 70 Chinese patients (59 men, 11 women) with primary esophageal squamous cell carcinomas (20 well-differentiated, 36 moderately differentiated, and 14 poorly differentiated squamous cell carcinomas) were tested for the presence of HPV and p53 mutation using the polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing. No HPV type 18 was detected, whereas HPV type 16 was identified in 8.6% (6 of 75) of the cases. p53 mutation was found in 44% (31 of 70) of the tumors. The mean ages of HPV-positive and HPV-negative groups of patients were 55 and 64 years, respectively (P = .046, t-test). There was no correlation between the prevalence of HPV and p53 mutation in these tumors. The presence of HPV and p53 also had no relation to the sex of the patients or to the grade of the carcinomas. It is concluded that the overall low prevalence of HPV in esophageal carcinomas may suggest that the virus may not play an important role in the pathogenesis of these tumors in Hong Kong Chinese patients. Also, p53 mutation and integrated HPV DNA are not mutually exclusive in esophageal cancer.