Microcell Hybrids Research Articles

Human artificial chromosome carrying 3p21.3-p22.2 region suppresses hTERT transcription in oral cancer cells

Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer

Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes.

Induction of genetic instability by transfer of a UV-A-irradiated chromosome

Exposure of cells to ultraviolet (UV)-A radiation induces oxidative damage in DNA, such as 8-oxo-7,8-dihydroguanine (8-oxoG), single-strand breaks, a-basic sites, and DNA–protein cross-links, via reactions with reactive oxygen species (ROS). In this study we examine whether the damage other than double-strand breaks (non-DSB damage), which is UV-A-induced oxidative damage, plays a role in the induction of chromosomal instability. We exposed human chromosome 21 to UV-A and transferred the chromosome into non-irradiated mouse recipient cells by microcell-mediated chromosome transfer. The chromosomal instability of both the transferred human chromosome and the recipient mouse chromosomes was examined by whole-chromosome painting and fluorescence in situ hybridization (WCP-FISH). The ploidy of the mouse recipient cells increased, and chromosomal aberrations occurred not only in the UV-A-irradiated human chromosome but also in the non-irradiated mouse chromosomes. The frequencies of these abnormalities increased with the radiation dose received by the transferred human chromosome. In contrast, in the control experiment, in which an non-irradiated human chromosome was transferred, the micro-cell hybrids remained diploid, and the frequency of chromosomal aberrations in both the transferred human chromosome and recipient mouse chromosomes remained low. Thus, the present study indicates that a chromosome harboring non-DSB damage induced by UV-A irradiation is unstable and transmits instability to chromosomes of non-irradiated recipient mouse cells.

The transfer of human artificial chromosomes via cryopreserved microcells

Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at -80°C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Abstract 3998: The role of serum amyloid A1 (SAA1) in nasopharyngeal carcinoma (NPC)

Abstract Background and aims: Nasopharyngeal carcinoma (NPC) is a cancer which occurs in high frequency in Southern China. The identification of diagnostic and prognostic markers will be highly beneficial for early stage NPC detection. Previous microcell-mediated chromosome transfer (MMCT) studies showed that the transfer of an intact human chromosome 11 suppresses the in vivo tumor growth of a NPC cell line (HONE1) in nude mouse tumorigenicity assays. We aim to identify candidate genes which are associated with tumor suppression in NPC. Methods: Differential expression analysis of 19K genes was performed in oligonucleotide microarray hybridization for the chromosome 11 microcell hybrids (MCHs)/ tumor segregants (TSs) pairs to hunt for some other unknown NPC genes. The gene expression of the candidate gene was examined in the NPC cell lines and tumor tissues. Functional analysis of restoration of the candidate gene expression was studied. Results: Using oligonucleotide microarray analysis, Serum Amyloid A 1 (SAA1), mapping close to 11p15.1, was identified as showing consistent down-regulated expression in the TSs, as compared to their parental tumor-suppressing MCHs. Gene expression and protein analyses show that SAA1 was not expressed in the NPC HONE1 recipient cells, tumor segregants, and other NPC cell lines; SAA1 was exclusively expressed in the non-tumorigenic MCHs. The mechanism of SAA1 gene inactivation in these NPC cell lines was attributed to hypermethylation. The clinical relevance of SAA1 in NPC was examined by RT-PCR; 54.8% (23/42) of NPC specimens showed either down-regulation or loss of SAA1 gene expression. After transfection of SAA1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. Conclusions: These findings suggest that SAA1 is a candidate tumor suppressor gene in NPC. Acknowledgments Grant support: Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China grant HKU772309 (H.L. Lung) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3998. doi:1538-7445.AM2012-3998

Identification of PITX1 as a TERT Suppressor Gene Located on Human Chromosome 5

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

The ECM protein LTBP‐2 is a suppressor of esophageal squamous cell carcinoma tumor formation but higher tumor expression associates with poor patient outcome

Our previous studies of chromosome 14 transfer into tumorigenic esophageal squamous cell carcinoma (ESCC) cell line, SLMT, suggested the existence of tumor suppressor genes on chromosome 14. Gene expression profiling of microcell hybrids and the tumor segregants identified an interesting gene, LTBP-2 (latent transforming growth factor β binding protein 2), which has been analyzed here for its role in ESCC. LTBP-2 maps to 14q24 and encodes a secreted protein, which is a component of the extracellular matrix microfibrils. LTBP-2 expression was downregulated in ESCC cell lines and tumor tissues. Promoter hypermethylation was found to be involved in LTBP-2 inactivation. Functional studies indicated its tumor-suppressive roles in ESCC. In the in vitro colony formation and Matrigel three-dimensional culture assays, LTBP-2 decreased the colony-forming abilities of ESCC cell lines. LTBP-2 expression was associated with reduction of cell migrating and invasive abilities. LTBP-2 could also reduce the tube-forming ability of endothelial cells. Moreover, LTBP-2 induced tumor suppression in in vivo nude mouse assays. Tissue microarray immunohistochemical staining analysis indicated that LTBP-2 expression is reduced in tumor tissues when compared to normal tissues, and LTBP-2 expression correlated significantly with the survival of ESCC patients. Thus, LTBP-2 appears to play an important role in ESCC.

Human chromosome 5 carries a transcriptional regulator of human telomerase reverse transcriptase ( hTERT)

Telomerase activation is crucial for cells that tend to be immortalized. Increased telomerase activity is correlated with upregulation of telomerase reverse transcriptase ( TERT) expression. In most human somatic cells, hTERT expression is suppressed by multiple factors. We have previously shown that human chromosome 5 carries a possible suppressor of mouse tert mtert expression in a mouse melanoma cell line, B16-F10 cells. However, the function of the transcriptional regulator of TERT on this chromosome remains unclear. To examine the functional role of a putative hTERT regulator(s) on this chromosome, we transferred human chromosome 5 in a human melanoma cell line, A2058 cells by microcell-mediated chromosome transfer (MMCT). Microcell hybrid clones with an introduced chromosome 5, but not chromosome 10, showed a remarkable decrease in the growth rate with an obvious cellular morphological alteration and eventually cellular senescence. Moreover, this phenomenon was accompanied by a reduction of hTERT expression and telomerase activity. Most importantly, we found that transcriptional suppression of hTERT by the introduction of chromosome 5 is largely mediated by regulating hTERT promoter activity. Furthermore, the hTERT promoter region between −1623 and −1047 was responsible for this function. These results provide evidence that transcriptional regulator(s) of the hTERT is carried on human chromosome 5 as an endogenous mechanism of hTERT suppression.

Exploitation of the interaction of measles virus fusogenic envelope proteins with the surface receptor CD46 on human cells for microcell-mediated chromosome transfer

BackgroundMicrocell-mediated chromosome transfer (MMCT) is a technique by which a chromosome(s) is moved from donor to recipient cells by microcell fusion. Polyethylene glycol (PEG) has conventionally been used as a fusogen, and has been very successful in various genetic studies. However, PEG is not applicable for all types of recipient cells, because of its cell type-dependent toxicity. The cytotoxicity of PEG limits the yield of microcell hybrids to low level (10-6 to 10-5 per recipient cells). To harness the full potential of MMCT, a less toxic and more efficient fusion protocol that can be easily manipulated needs to be developed.ResultsMicrocell donor CHO cells carrying a human artificial chromosome (HAC) were transfected with genes encoding hemagglutinin (H) and fusion (F) proteins of an attenuated Measles Virus (MV) Edmonston strain. Mixed culture of the CHO transfectants and MV infection-competent human fibrosarcoma cells (HT1080) formed multinucleated syncytia, suggesting the functional expression of the MV-H/F in the CHO cells. Microcells were prepared and applied to HT1080 cells, human immortalized mesenchymal stem cells (hiMSC), and primary fibroblasts. Drug-resistant cells appeared after selection in culture with Blasticidin targeted against the tagged selection marker gene on the HAC. The fusion efficiency was determined by counting the total number of stable clones obtained in each experiment. Retention of the HAC in the microcell hybrids was confirmed by FISH analyses. The three recipient cell lines displayed distinct fusion efficiencies that depended on the cell-surface expression level of CD46, which acts as a receptor for MV. In HT1080 and hiMSC, the maximum efficiency observed was 50 and 100 times greater than that using conventional PEG fusion, respectively. However, the low efficiency of PEG-induced fusion with HFL1 was not improved by the MV fusogen.ConclusionsEctopic expression of MV envelope proteins provides an efficient recipient cell-oriented MMCT protocol, facilitating extensive applications for studies of gene function and genetic corrections.

Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma

THY1 was previously identified as a candidate tumor suppressor gene (TSG) associated with lymph node metastases in nasopharyngeal carcinoma (NPC) through functional studies. It was identified by oligonucleotide microarray analysis as an interesting differentially expressed gene. However, direct functional evidence is still lacking for THY1 being a TSG in NPC, as in vivo tumorigenicity assays have not been previously reported in our last study of THY1. In this study, a tetracycline-inducible expression vector, pETE-Bsd, was used to obtain stable transfectants of THY1. The stringent in vivo tumorigenicity assay results show that the activation of THY1 suppresses tumor formation of HONE1 cells in nude mice, and the tumor formation ability was restored in the presence of doxycycline (a tetracycline analog), when the gene is shut off. Functional inactivation of this gene is observed in all the tumors derived from the tumorigenic transfectant. The tumor suppressive effect could be repressed by knockdown of THY1 expression in nontumorigenic microcell hybrids. Further studies indicate that expression of THY1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)/G(1) phase. It greatly reduces the ability for anchorage-independent growth. The invasiveness of HONE1 cells was also inhibited by the expression of THY1. These findings suggest that THY1 is a TSG in NPC, which is involved in invasion and shows an association with tumor metastasis. Taken together, THY1 clearly plays an important functional role in tumor suppression in NPC.

Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, Mirror image polydactyly 1 , in nasopharyngeal carcinoma

Chromosome 14 allelic loss is common in nasopharyngeal carcinoma (NPC) and may reflect essential tumor suppressor gene loss in tumorigenesis. An intact chromosome 14 was transferred to an NPC cell line using a microcell-mediated chromosome transfer approach. Microcell hybrids (MCHs) containing intact exogenously transferred chromosome 14 were tumor suppressive in athymic mice, demonstrating that intact chromosome 14 NPC MCHs are able to suppress tumor growth in mice. Comparative analysis of these MCHs and their derived tumor segregants identified 4 commonly eliminated tumor-suppressive CRs. Here we provide functional evidence that a gene, Mirror-Image POLydactyly 1 (MIPOL1), which maps within a single 14q13.1-13.3 CR and that hitherto has been reported to be associated only with a developmental disorder, specifically suppresses in vivo tumor formation. MIPOL1 gene expression is down-regulated in all NPC cell lines and in approximately 63% of NPC tumors via promoter hypermethylation and allelic loss. SLC25A21 and FOXA1, 2 neighboring genes mapping to this region, did not show this frequent down-regulated gene expression or promoter hypermethylation, precluding possible global methylation effects and providing further evidence that MIPOL1 plays a unique role in NPC. The protein localizes mainly to the nucleus. Re-expression of MIPOL1 in the stable transfectants induces cell cycle arrest. MIPOL1 tumor suppression is related to up-regulation of the p21(WAF1/CIP1) and p27(KIP1) protein pathways. This study provides compelling evidence that chromosome 14 harbors tumor suppressor genes associated with NPC and that a candidate gene, MIPOL1, is associated with tumor development.

The functional evidence for metastasis suppressor genes of liver cancer harboring on chromosome 8

Objective To find out the functional evidence for metastasis suppressor gene in liver cancer on human chromosome 8p. Methods The microcell hybrid clones Neo8/C5F-1-10, which were generated by introducing human chromosome 8 randomly marked with neo gene into C5F cell line by mi-crocell mediated chromosome transfer (MMCT) and the C5F cell line were subcutaneously implanted in 6-7 week-old female BALB/CA nude mice to perform the spontaneous metastasis assay. Serial dissections of the lung tissues were done to count the microscopic metastatic foci. The number of the metastatic foci was compared by one-way ANOVA. Results All the microceli hybrids implanted had grown into measurable tumors subcutaneously. The spontaneous metastasis assay for these microcell hybrid clones showed that 6 clones displayed prominent lung metastasis suppression (P<0.05). Among 2 of them, lung metastases had been totally suppressed. Conclusion The successful introduction of human chromosome 8 into meta-static rat liver cancer cell line had changed the metastatic behavior of C5F, which provided the direct func-tional evidence for the metastasis suppressor gene in liver cancer harboring on human chromosome 8. Key words: Carcinoma,hepatocellular; Chromosomes; Metastasis; Suppressor genes

Functional Analysis of a Cell Cycle–Associated, Tumor-Suppressive Gene, Protein Tyrosine Phosphatase Receptor Type G, in Nasopharyngeal Carcinoma

Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis.

Characterization of a novel epigenetically‐silenced, growth‐suppressive gene, ADAMTS9, and its association with lymph node metastases in nasopharyngeal carcinoma

AbstractBy using a functional complementation approach, suppression of tumorigenicity was observed after transfer of intact or truncated copies of chromosome 3 into a nasopharyngeal carcinoma (NPC) HONE1 cell line. The extra exogenous chromosome 3 in the microcell hybrids (MCHs) significantly extended the lag period of tumor formation, which may be associated with loss or inactivation of wild type alleles from the normal donor chromosome 3. Representative tumors, which grew in nude mice were reconstituted into culture and expanded as tumor segregants (TSs). In our study, a disintegrin‐like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9), a gene mapping to 3p14.2, was identified to be critically associated with tumor suppression in NPC. Gene expression analysis showed that ADAMTS9 was either not expressed or was downregulated in HONE1 cells, TSs and NPC cell lines. The mechanism of ADAMTS9 gene inactivation in the NPC cell lines and tissues was attributed to promoter hypermethylation. Using a tissue microarray and immunohistochemical staining, 31 of 66 (47%) of the NPC cases showed downregulated or absence of ADAMTS9 expression. ADAMTS9 expression was downregulated or lost in 17 of 23 (73.9%) lymph node metastatic NPC specimens, which was significantly higher than in 14 of 43 (32.6%) primary tumors. After transfection of the ADAMTS9 gene into 7 NPC cell lines, a dramatic reduction of colony forming ability was observed. These findings support ADAMTS9 as a putative tumor suppressor gene in vivo in NPC that is significantly associated with lymph node metastases. © 2008 Wiley‐Liss, Inc.

Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and THSD1 in Esophageal Carcinoma

Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG.

Re-expression of tumorigenicity after attenuation of human synaptotagmin 13 in a suppressed microcell hybrid cell line

Human chromosome 11p11.2 contains a putative liver tumor suppressor locus that was identified using a microcell hybrid cell line-based model of tumor suppression. Transcription mapping of suppressed microcell hybrid cell lines suggests that human SYT13 represents a strong candidate for the 11p11.2 tumor suppressor gene. Other evidence suggests that the putative 11p11.2 tumor suppressor gene (SYT13 or some other) may modulate the tumorigenic potential of neoplastic liver cell lines through direct or indirect regulation of the rat Wt1 tumor suppressor gene. To characterize a functional role for SYT13 in liver tumor suppression, we employed RNAi to attenuate SYT13 expression in a suppressed microcell hybrid cell line (GN6TF-11neoCX4). SYT13-attenuated cells display aggressive phenotypic properties that are similar to or indistinguishable from the parental tumor cells (GN6TF), including altered cellular morphologies, disrupted contact inhibition, elevated saturation densities, restoration of anchorage-independent growth and increased tumorigenicity in vivo. Moreover, SYT13 attenuation and re-expression of tumorigenicity in GN6TF-11neoCX4-derived cell lines was accompanied by a significant decrease of Wt1 expression. In contrast, the phenotypic properties of scrambled-control cells were similar to the suppressed microcell hybrid cells and Wt1 expression was unaffected. These observations combine to establish that: i) human SYT13 functions as a liver tumor suppressor gene that complements a molecular defect in GN6TF rat liver tumor cells resulting in a normalized cellular phenotype in vitro and suppression of tumorigenicity in vivo; ii) RNAi-mediated attenuation of SYT13 expression restores the neoplastic phenotype of GN6TF-11neoCX4 microcell hybrid cells, consistent with the function of a liver tumor suppressor gene; and iii) loss of Wt1 expression is important for the re-establishment of tumorigenic potential by GN6TF-11neoCX4 microcell hybrid cells after attenuation of SYT13.

Re-expression of tumorigenicity after attenuation of human synaptotagmin 13 in a suppressed microcell hybrid cell line

Re-expression of tumorigenicity after attenuation of human synaptotagmin 13 in a suppressed microcell hybrid cell line