Biomedical subjects
Publications and source records attributed to Sai Wah Tsao.
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Chromosome 3p plays an important role in tumorigenesis in many cancers, including nasopharyngeal carcinoma (NPC). We have previously shown chromosome 3p can suppress tumor growth in vivo by using the monochromosome transfer approach, which indicated the chromosome 3p21.3 region was critical for tumor suppression. BLU/ZMYND10 is one of the candidate tumor suppressor genes mapping in the 3p21.3 critical region and is a candidate TSG for NPC. By quantitative RT-PCR, it is frequently downregulated in NPC cell lines (83%) and NPC biopsies (80%). However, no functional studies have yet verified the functional role of BLU/ZMYND10 as a tumor suppressor gene. In the current study, a gene inactivation test (GIT) utilizing a tetracycline regulation system was used to study the functional role of BLU/ZMYND10. When BLU/ZMYND10 is expressed in the absence of doxycycline, the stable transfectants were able to induce tumor suppression in nude mice. In contrast, downregulation of BLU/ZMYND10 in these tumor suppressive clones by doxycycline treatment restored the tumor formation ability. This study provides the first significant evidence to demonstrate BLU/ZMYND10 can functionally suppress tumor formation in vivo and is, therefore, likely to be one of the candidate tumor suppressor genes involved in NPC.
EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH(2)-terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IkappaB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor kappaB and p38/mitogen-activated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1.
OBJECTIVE: To evaluate the effect of S-allylcysteine (SAC) on CWR22R, a human androgen-independent (AI) prostate cancer xenograft, in nude mice. Despite extensive research worldwide there is no effective way to control the growth of prostate cancer, and we previously reported that SAC and S-allylmercaptocysteine (SAMC), two water-soluble derivatives of garlic, inhibit cancer cell invasion through restoration of E-cadherin expression in vitro. MATERIALS AND METHODS: The effects of SAC on tumour cell proliferation markers such as Ki-67 and proliferating cell nuclear antigen, and apoptotic regulators including Bcl-2 and cleaved caspase-3, were assessed by immunohistochemical staining. The inhibitory effects of SAC on prostate cancer invasion was examined by immunoreactivity of E-cadherin and its binding proteins alpha, beta and gamma-catenins. The serum prostate-specific antigen (PSA) level at three different times (initiation, middle and end of treatment) and toxicity of SAC on several organs after treatment were assessed. RESULTS: Treatment with SAC resulted in inhibition of the growth of CWR22R, with no detectable toxic effect on nude mice. The SAC-induced growth reduction was correlated with a concurrent reduction in serum PSA level and proliferation rate of xenografts, together with an inhibition of invasion through the restoration of E-cadherin and gamma-catenin expression. Furthermore, the apoptotic rate of SAC-treated tumours increased together with a decrease in Bcl-2 and increase in cleaved caspase-3. CONCLUSION: These results suggest that this garlic-derived compound might be a potential therapeutic agent for suppressing AI prostate cancer.
In up to 87% of nasopharyngeal carcinoma (NPC) clinical tumor specimens, there was either down-regulation or loss of TSLC1 gene expression. Using a tissue microarray and immunohistochemical staining, the frequency of down-regulated or loss of expression of TSLC1 in metastatic lymph node NPC was 83% and the frequency of loss of expression of TSLC1 was 35%, which was significantly higher than that in primary NPC (12%). To examine the possible growth-suppressive activity of TSLC1 in NPC, three NPC cell lines, HONE1, HNE1, and CNE2, were transfected with the wild-type TSLC1 gene cloned into the pCR3.1 expression vector; a reduction of colony formation ability was observed for all three cell lines. A tetracycline-inducible expression vector, pETE-Bsd, was also used to obtain stable transfectants of TSLC1. There was a dramatic difference between colony formation ability in the presence or absence of doxycycline when the gene is shut off or expressed, respectively, with the tetracycline-inducible system. Tumorigenicity assay results show that the activation of TSLC1 suppresses tumor formation in nude mice and functional inactivation of this gene is observed in all the tumors derived from tumorigenic transfectants. Further studies indicate that expression of TSLC1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)-G(1) phase in normal culture conditions, whereas in the absence of serum, TSLC1 induced apoptosis. These findings suggest that TSLC1 is a tumor suppressor gene in NPC, which is significantly associated with lymph node metastases.
The helix-loop-helix protein inhibitor of differentiation and DNA binding (Id-1) is known to promote cellular proliferation in several types of human cancer. Although it has been reported that Id-1 is over-expressed in esophageal squamous cell carcinoma (ESCC), its function and signaling pathways in esophageal cancer are unknown. In our study, we investigated the direct effects of Id-1 on esophageal cancer cell growth by transfecting an Id-1 expression vector into an ESCC cell line (HKESC-3), which showed serum-dependent Id-1 expression. Ectopic Id-1 expression resulted in increased serum-independent cell growth and G1-S phase transition, as well as up-regulation of mouse double minute 2 (MDM2) and down-regulation of p21Waf1/Cip1 protein expressions in the transfectant clones in a p53-independent manner. However, overexpression of Id-1 had no effect on the pRB, CDK4 and p16INK4A expressions. Stable transfection of Id-1 antisense expression vector to inhibit the expression of endogenous Id-1 in another ESCC cell line (HKESC-1) reversed the effects on MDM2 and p21Waf1/Cip1. In addition, Id-1 expression protected ESCC cells from Tumor Necrosis Factor (TNF)-alpha-induced apoptosis by up-regulating and activating Bcl-2. In conclusion, our study provides evidence for the first time that Id-1 plays a role in both proliferation and survival of esophageal cancer cells. Our findings also suggest that unlike prostate, hepatocellular and nasopharyngeal carcinomas in which Id-1 induces cell proliferation through inactivation of p16INK4A/RB pathway, the increased cell proliferation observed in ESCC cells may be mediated through a different mechanism.
Metastatic cancer is one of the main causes of cancer-related death since they rarely respond to available treatments. Recently, certain compounds isolated from the dietary supplement, garlic, have shown anti-proliferation effect on cancer cells. The aim of this study was to investigate whether certain garlic derivatives had any effect on the potentially invasive androgen-independent prostate cancer (PCa) cells. Using colony-forming, wound-closure as well as matrigel-invasion assays, we found that two main water-soluble constituents of the garlic, S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC), were able to suppress PCa cell proliferation and invasive abilities. This inhibitory effect was associated with induction of mesenchymal to epithelial transition. Most importantly, the SAC and SAMC treatment led to restoration of E-cadherin expression at transcription and protein levels. In contrast, the expression of E-cadherin repressor, Snail, was reduced in the SAC- and SAMC-treated cells. Furthermore, examination of cell lines from other types of cancer (ovarian, nasopharyngeal and esophageal carcinomas) also confirmed that the effect of SAC and SAMC on activation of E-cadherin might be a general effect on human cancer cells. Our results demonstrate a novel anticancer effect of garlic and suggest that certain garlic-derived compounds may be potential agents for suppression of invasive growth through restoration of E-cadherin expression in cancer cells.
Rev7p has been suggested to play an important role in regulating DNA damage response in yeast, and recently, the human homologue (i.e., MAD2B) has been identified, which shares significant homology to the mitotic checkpoint protein MAD2. In this study, we investigated whether MAD2B played a key role in cellular sensitivity to DNA-damaging anticancer drugs by suppressing its expression using RNA interference in nasopharyngeal carcinoma cells. Using colony formation assay, we found that suppression of MAD2B conferred hypersensitivity to a range of DNA-damaging agents, especially DNA cross-linkers, such as cisplatin, and gamma-irradiation. This effect was associated with reduced frequencies of spontaneous and drug-induced mutations, elevated phosphorylation of histone H2AX, and markedly increased chromosomal aberrations in response to DNA damage. In addition, there was also a significant decrease in cisplatin-induced sister chromatid exchange rate, a marker for homologous recombination-mediated post-replication repair in MAD2B-depleted cells. These results indicate that MAD2B may be a key factor in regulating cellular response to DNA damage in cancer cells. Our findings reveal a novel strategy for cancer therapy, in which cancer cells are sensitized to DNA-damaging anticancer drugs through inactivation of the MAD2B gene.
BACKGROUND INFORMATION: Loss of sensitivity to TGF-beta1 (transforming growth factor beta1)-induced growth arrest is an important step towards malignant transformation in human epithelial cells, and Id-1 (inhibitor of differentiation or DNA binding-1) has been associated with cell proliferation and cell-cycle progression. Here, we investigated the role of Id-1 in cellular sensitivity to TGF-beta1. RESULTS: Using an immortalized prostate epithelial cell line, NPTX cells, we suppressed Id-1 expression through antisense strategy. We found that inhibition of Id-1 expression suppressed cell proliferation and at the same time induced cellular senescence and G2/M cell-cycle arrest. In addition, inactivation of Id-1 made cells more vulnerable to TGF-beta1-induced growth arrest. The sensitization effect on TGF-beta1 was associated with up-regulation of two downstream effectors of the TGF-beta1 pathway, p21WAF1/Cip1 and p27KIP1. CONCLUSION: Our results indicate that endogenous Id-1 levels might be a crucial factor in the development of resistance to TGF-beta1-induced growth suppression in human prostate epithelial cells.
Epstein-Barr virus (EBV) latent infection is a critical event in nasopharyngeal carcinoma (NPC) tumorigenesis. EBV-encoded genes have been shown to be involved in immune evasion and in the regulation of various cellular signaling cascades. To elucidate the roles of EBV in NPC development, stable infection of EBV in nasopharyngeal epithelial cell lines was established. Similar to primary tumors of NPC, these infected cells exhibited a type II EBV latency expression pattern. In this study, multiple cellular signaling pathways in EBV-infected cells were investigated. We first demonstrated that in vitro EBV infection resulted in the activation of STAT3 and NFkappaB signal cascades in nasopharyngeal epithelial cells. Increased expression of their downstream targets (c-Myc, Bcl-xL, IL-6, LIF, SOCS-1, SOCS-3, VEGF, and COX-2) was also observed. Moreover, EBV latent infection induced the suppression of p38-MAPK activities, but did not activate PKR cascade. Our findings suggest that EBV latent infection is able to manipulate multiple cellular signal cascades to protect infected cells from immunologic attack and to facilitate cancer development.
Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.
Recently, mitotic arrest deficient 2 (MAD2)-mediated spindle checkpoint is shown to induce mitotic arrest in response to DNA damage, indicating overlapping roles of the spindle checkpoint and DNA damage checkpoint. In this study, we investigated if MAD2 played a part in cellular sensitivity to DNA-damaging agents, especially cisplatin, and whether it was regulated through mitotic checkpoint. Using nine nasopharyngeal carcinoma (NPC) cell lines, we found that decreased MAD2 expression was correlated with cellular resistance to cisplatin compared with the cell lines with high levels of MAD2. Exogenous MAD2 expression in NPC cells also conferred sensitivity to DNA-damaging agents especially cisplatin but not other anticancer drugs with different mechanisms of action. The increased cisplatin sensitivity in MAD2 transfectants was associated with mitotic arrest and activation of apoptosis pathway evidenced by the increased mitotic index and apoptosis rate as well as decreased Bcl-2 and Bax ratio and expression of cleaved poly(ADP-ribose) polymerase and caspase 3. Our results indicate that the MAD2-induced chemosensitization to cisplatin in NPC cells is mediated through the induction of mitotic arrest, which in turn activates the apoptosis pathway. Our evidence further confirms the previous hypothesis that spindle checkpoint plays an important part in DNA damage-induced cell cycle arrest and suggests a novel role of MAD2 in cellular sensitivity to cisplatin.
Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers. Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. Epigenetic inactivation of CHFR through promoter CpG hypermethylation may lead to CIN and has been reported in several human cancers. In this study, we investigated the CHFR gene expression in a panel of nasopharyngeal carcinoma (NPC), prostate, ovarian, and breast cancer cell lines. We found that the expression of CHFR mRNA was significantly decreased or undetectable in all eight NPC cell lines as well as three human NPC xenografts, whereas non-malignant nasopharyngeal cell lines and other cancer cell lines tested expressed CHFR at relatively high levels. Hypermethylation of CHFR promoter region was also strongly correlated with decreased CHFR expression in NPC cell lines and xenografts. Treatment with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, led to restoration of CHFR expression in NPC cell lines. More importantly, hypermethylation of CHFR promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non-malignant tissues. These findings suggest that downregulation of CHFR is a common event in NPC cells which may be due to hypermethylation of the gene promoter region.
Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.
Extravillous cytotrophoblast (EVCT) cultures from the normal placentas of three pregnant women were transfected by HPVE6E7. Sequential cytogenetic and molecular analyses were performed to delineate genetic events that may be critical for cell immortalization. One line, PE1-E6E7, was immortalized successfully, whereas 2 other lines, PE3-E6E7 and PE4-E6E7, could not be maintained beyond crisis. Before crisis, the majority of cells in all lines were karyotypically normal. During the early stages of crisis, there was progressive telomere shortening. Most cells were karyotypically abnormal, with extreme cytogenetic divergence and a predominance of telomeric association and dicentric chromosomes affecting many chromosomes. At the later stages of crisis, the karyotype became more convergent with a drastic decrease in nonclonal aberrations. In PE1-E6E7, after crisis the karyotype was complex, with frequent centromeric rearrangements in the form of isochromosomes and whole-arm translocations. There were unbalanced structural aberrations and numerical changes, including loss of chromosome 13, that could be traced throughout the evolution of the line. These findings support the concept that immortalization is a relatively rare and nonrandom event that occurs only in cells that have acquired the necessary or critical genetic alterations. Telomeric dysfunction may be an important mechanism leading to the acquisition of complex karyotypical aberrations.
BACKGROUND: Infection with high-risk human papillomavirus (HPV) has been implicated as one of the risk factors of esophageal squamous cell carcinoma (ESCC). Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of the HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. OBJECTIVES AND STUDY DESIGN: To investigate the viral integration status of HPV-16 infection in ESCC, 35 HPV-positive ESCC specimens collected from Chinese patients were subject to real-time quantitative PCR for determination of physical status of HPV-16 by analyzing the ratios of E2 to E6 genes. RESULTS: Our results showed that only 8.6% (3/35) of the HPV-16 positive specimens harbored exclusively the episomal form (i.e. E2/E6 ratio > or = 1), whereas the remaining 91.4% contained either only the integrated form (5.7%, with E2/E6 ratio = 0) or a mixture of episomal and integrated forms of viral molecules (85.7%, with E2/E6 ratios > 0 but < 1). Amongst the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/integrated E6 ratios of less than 1, indicating a predominance of integrated form of viral genes in these lesions. CONCLUSION: Our finding of frequent integration of viral DNA in the host genome suggests that integration HPV-16 is common in ESCC from Chinese patients and implies that HPV infection may play a role in the pathogenesis of ESCC.
OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.
Chromosomal aberrations are common in cancers. However, the search for chromosomal aberrations leading to development of specific solid tumors has been severely hindered because the majority of solid tumors have complex chromosomal aberrations that differ within the same tumor types. A similar phenomenon exists in immortalized cell lines. The underlying mechanisms driving these diverse aberrations are largely unknown. Telomeres play crucial roles in protecting the integrity of eucaryotic chromosomes and maintaining genomic stability of human cells. Telomere lengths on individual chromosomes in normal human somatic cells are heterogeneous and undergo progressive shortening with aging process. In this study, for the first time, a molecular cytogenetic method using sequential telomere quantitative fluorescence in situ hybridization and spectral karyotyping on the same human metaphases was applied successfully to examine the dynamic profiles of individual telomere shortening and their relationship to chromosome aberrations in multiple human cell lines undergoing immortalization. Human ovarian surface epithelial cells and esophageal epithelial cells were immortalized by the expression of HPV16 E6 and E7, which drive cells to proliferate by inactivating p53 and Rb genes. In these cell lines, we consistently detected large-scale differences in telomere signal intensities not only among nonhomologous chromosome arms but also between some homologous chromosome arms. The cell lines derived from different donors had different profiles of critically short telomeres (lacking telomere signals). Strikingly, the different profiles of chromosomal structural aberrations in multiple immortalized cell lines were highly significantly associated with the distinct distributions of critically short telomeres in whole-genome. Since cellular immortalization is one of the hallmarks of cancer, our findings suggest that distinct profiles of critically short telomeres in different human individuals might play an essential role in determining the complex and individual-specific chromosomal structural aberrations in human solid tumors.