Identification of tumor suppressive activity by irradiation microcell-mediated chromosome transfer and involvement of alpha B-crystallin in nasopharyngeal carcinoma.

In previous studies, we successfully refined nasopharyngeal carcinoma (NPC) critical regions (CRs) mapping to chromosome 11q13 and 11q22-23. The chromosome 11 fragment containing the 1.8 Mb NPC CR at 11q13 (CR1), the CR at 11q22.3 mapped near D11S2000 (CR2), part of the CR at 11q23.1-11q23.2 overlapping with D11S1300 and D11S1391 (CR3), and the CR at cell adhesion molecule 1 (CADM1) locus (CR4), was chosen as the chromosome 11 donor cell line for the present study. Gamma irradiation was applied to cleave this truncated chromosome into smaller fragments and a new panel of donor cells containing further deleted fragments was produced. Subclones XMCH3.2 and XMCH3.4 were chosen for subsequent transfer to HONE1 cells; each contains a single copy of deleted chromosome 11 fragment with or without CR2 and the THY1 locus, previously shown to be involved in NPC. Both resultant chromosome 11 fragments in XMCH3.2 and XMCH3.4 caused tumor suppression. The association of alpha B-crystallin (CRYAB), a gene identified as being differentially expressed by gene profiling of NPC and an immortalized nasopharyngeal epithelial cell line, and which is located near CR3, was found to be associated with tumor suppression in all the tumor-suppressive hybrids. In addition, the expression level of this gene was down-regulated in the 7 NPC cell lines and in 5 out of 14 normal/tumor tissue pairs in the present study. Both promoter hypermethylation and allelic loss may be involved in the inactivation of this gene, suggesting its possible role in NPC development.

Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma.

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.

Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis.

Although the integration of hepatitis B virus (HBV) into human DNA has been found to be associated with the development of hepatocellular carcinoma (HCC), the molecular mechanism remains unclear. In order to obtain additional insight into the correlation of HBV integration and HCC development, integrated HBV in 14 primary HCC cases was isolated and characterized by sequencing analysis. Our findings in this study showed that: (1) none of the known cellular oncogene or tumor suppressor gene was affected by the HBV integration; (2) although the integration of HBV is random, the integration site was often within or close to human repetitive sequences; (3) integrated HBV may possess the capacity to transpose to another chromosome region through reintegration; (4) rearrangements of HBV sequence were observed in all the 14 integrants, involving (most frequently) X (12/14 integrants), P (8/14), S (7/14), and C (7/14) genes; and (5) 3'-deleted X gene and consequent C-terminal truncated X protein caused by HBV integration was observed in 10 cases. These deletions cause the losses of p53-dependent transcriptional repression binding site, transcription factor Sp1 binding site, and growth-suppressive effect domain, leading to cell proliferation and transformation. This finding suggests that 3'-deleted X gene caused by the HBV integration may play an important role in the HCC development.

Establishment and characterization of a human non-small cell lung cancer cell line.

Lung cancer is one of the most common causes of cancer death worldwide. Although many efforts have been made to explore the mechanisms involved in the development of lung cancer, the genetic events involved in the pathogenesis of lung cancer are still unclear. For a better mechanistic scope of study, a well-established cellular model is essential. We report the establishment of a squamous cell carcinoma (SCC) cell line of human lung, SCC-37. Chromosomal abnormalities and global genomic alterations of SCC-37 were studied by spectral karyotyping (SKY) and comparative genomic hybridization (CGH), respectively. Results showed that SCC-37 was a hypodiploid with complex chromosomal rearrangements. Some of the alterations, such as the gain of 1q25-qter in SCC-37, have been correlated to the tumor recurrence of non-small cell lung cancer (NSCLC). Other interesting findings include the amplification of 3q25-qter and 12q13, suggesting the existence of important oncogenes in the amplicons. This cell line may thus provide a useful cellular resource for studying the pathogenesis of SCC of the lung in the future.

Quantitative plasma hypermethylated DNA markers of undifferentiated nasopharyngeal carcinoma.

PURPOSE: Gene-specific methylation is common in primary undifferentiated nasopharyngeal carcinoma (NPC). DNA released from apoptotic or necrotic cell death including those aberrantly methylated promoter DNA of cancer cells is absorbed into the circulation as cell-free plasma DNA of the patient. This study aims at evaluation of the potential use of methylated gene promoter DNA as a serological tumor marker of primary and potentially salvageable local or nodal recurrent NPC. EXPERIMENTAL DESIGN: The quantity of plasma hypermethylated gene promoters of CDH1, DAPK1, p15, p16, RASSF1A, and MLH1 of 41 NPC patients before treatment and 43 normal individuals were studied using real-time quantitative PCR. The post-treatment plasma hypermethylated CDH1, DAPK1,and p16 were also measured in 13 NPC patients with locoregional recurrence and 17 patients in remission. RESULTS: Concentrations of cell-free circulating DNA were significantly higher in NPC patients than normal controls (28.79 ng/ml versus 16.57 ng/ml, respectively). There was no significant difference in plasma DNA concentration of EBV-positive and -negative normal individuals. Methylated DNA was detectable in plasma of NPC patients before treatment including 46% for CDH1,42% for p16,20% for DAPK1,20% for p15,and 5% for RASSF1A. Hypermethylated MLH1 was not detected in plasma of all of the NPC patients and normal individuals. Aberrantly hypermethylated promoter DNA of at least one of the five genes was detectable in 29 of 41 (71%) plasma of NPC patients before treatment. Hypermethylated promoter DNA of at least one of the three genes (CDH1, DAPK1, and p16) was detectable in post-treatment plasma of 5 of 13 (38%) recurrent NPC patients and none of the patients in remission. CONCLUSIONS: Our results suggested that cell-free circulating methylated gene promoter DNA is a possibly useful serological marker in assisting in screening of primary and potentially salvageable local or regional recurrent NPC.

Promoter hypermethylation of high-in-normal 1 gene in primary nasopharyngeal carcinoma.

PURPOSE: The methylation of high-in-normal-1 (HIN-1) gene promoter in undifferentiated nasopharyngeal carcinoma (NPC) is studied. EXPERIMENTAL DESIGN: The methylation status of HIN-1 in NPC cell lines, primary NPC, paired nasopharyngeal swabs, paired throat-rinsing fluid, and paired peripheral blood was assessed by methylation-specific PCR assay. The relationship between HIN-1 promoter methylation and transcription in NPC cell lines was evaluated by reverse transcription-PCR and demethylation agent treatment (5-aza-2-deoxycytidine). RESULTS: Hypermethylated promoter was observed in five of five (100%) NPC cell lines and not found in three normal nasopharyngeal outgrowths, two tonsil epithelial cell cultures, and two skin fibroblast cultures. Reverse transcription-PCR assay indicated that HIN-1 transcription was significantly down-regulated in the NPC cell line with promoter methylation. Treatment with demethylation agent, 5-aza-2-deoxycytidine, restored HIN-1 transcription in the NPC cell line. Methylated HIN-1 promoter was found in 36 of 47 (77%) primary NPC tumors and not found in the normal nasopharyngeal biopsies. Methylated HIN-1 promoter was detected in 12 of 26 (46%) nasopharyngeal swabs, 5 of 26 (19%) throat-rinsing fluids, 2 of 11 (18%) plasmas, and 5 of 11 (46%) buffy coats of peripheral blood of the NPC patients but was not detectable in all normal controls. CONCLUSION: HIN-1 promoter hypermethylation is common in NPC. Methylated promoter DNA in nasopharyngeal swab, throat-rinsing fluid, and peripheral blood might be potentially useful as tumor marker for screening of NPC.

Differential gene methylation in undifferentiated nasopharyngeal carcinoma.

Differential gene methylation is observed in a variety of human malignancies. The study of the pattern of methylated genes helps to understand carcinogenesis and to identify potential marker tumor genes for clinical use. The differential methylated genes in undifferentiated nasopharyngeal carcinoma (NPC) of Chinese were studied by methylation-specific polymerase chain reaction (MSP). Methylation status of 11 tumor-associated genes (ARF, caspase-8, CDH1, CDKN2A, CDKN2B, MGMT, MLH1, RASSF1A, THBS1, TP73 and VHL) was studied in 30 primary undifferentiated NPC and paired peripheral blood of 12 patients. The methylation profile of NPC in order of frequency was CDH1 (50%), CDKN2B (50%), THBS1 (50%), RASSF1A (46%), MLH1 (40%), MGMT (28%), CDKN2A (23%), TP73 (20%), caspase-8 (7%), ARF (3%) and VHL (0%). Methylation of at least 1 gene was observed in 93% of primary NPC. Of the 12 patients with at least 1 methylated gene in the primary tumor, all 12 (100%) patients had at least 1 of the methylated gene promoter detectable in their peripheral blood. The results show high frequency of methylation in NPC and the potential of using methylation as peripheral blood tumor marker in screening NPC.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry serum protein profiling to identify nasopharyngeal carcinoma.

BACKGROUND: Diagnosis of nasopharyngeal carcinoma (NPC) at an early disease stage is important for successful treatment and improving the outcome of patients. The use of serum protein profiles and a classification tree algorithm were explored to distinguish NPC from noncancer. METHODS: Serum samples were applied to metal affinity protein chips to generate mass spectra by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Protein peak identification and clustering were performed using the Biomarker Wizard software. Proteomic spectra of serum samples from 50 NPC patients and 54 noncancer controls were used as a training set and a classification tree with 6 distinct protein masses was generated by using Biomarker Pattern software. The validity of the classification tree was then challenged with a blind test set including another 20 NPC patients and 25 noncancer controls. RESULTS: The software identified an average of 93 mass peaks/spectrum and 6 of the identified peaks were used to construct the classification tree. The classification tree correctly determined 83% (123 of 149) of the test samples with 83% (58 of 70) of the NPC samples and 82% (65 of 79) of the noncancer samples. In a combination of the serum protein profiles with Epstein-Barr (EBV) nuclear antigen 1 (EBNA1 IgA) test, the diagnostic sensitivity and specificity were increased to 99% and 96%, respectively. CONCLUSIONS: The results suggest that SELDI-TOF-MS serum protein profiles could discriminate NPC from noncancer. The combination of serum protein profiles with an EBV antibody serology test could further improve the accuracy of NPC screening.

Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma.

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.

TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma.

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.