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.

A novel Xist RNA-mediated chromosome inactivation model using a mouse artificial chromosome.

OBJECTIVE: To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. RESULTS: Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. CONCLUSIONS: We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins.

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.

MTA1, a metastasis­associated protein, in endothelial cells is an essential molecule for angiogenesis.

Our previous study revealed that metastasis­associated protein 1 (MTA1), which is expressed in vascular endothelial cells, acts as a tube formation promoting factor. The present study aimed to clarify the importance of MTA1 expression in tube formation using MTA1­knockout (KO) endothelial cells (MTA1­KO MSS31 cells). Tube formation was significantly suppressed in MTA1­KO MSS31 cells, whereas MTA1­overexpression MTA1­KO MSS31 cells regained the ability to form tube­like structures. In addition, western blotting analysis revealed that MTA1­KO MSS31 cells showed significantly higher levels of phosphorylation of non­muscle myosin heavy chain IIa, which resulted in suppression of tube formation. This effect was attributed to a decrease of MTA1/S100 calcium­binding protein A4 complex formation. Moreover, inhibition of tube formation in MTA1­KO MSS31 cells could not be rescued by stimulation with vascular endothelial growth factor (VEGF). These results demonstrated that MTA1 may serve as an essential molecule for angiogenesis in endothelial cells and be involved in different steps of the angiogenic process compared with the VEGF/VEGF receptor 2 pathway. The findings showed that endothelial MTA1 and its pathway may serve as promising targets for inhibiting tumor angiogenesis, further supporting the development of MTA1­based antiangiogenic therapies.

Activation of PPARγ in bladder cancer via introduction of the long arm of human chromosome 9.

Bladder cancer is divided into two molecular subtypes, luminal and basal, which form papillary and nodular tumors, respectively, and are identifiable by gene expression profiling. Although loss of heterozygosity (LOH) of the long arm of human chromosome 9 (9q) has been observed in the early development of both types of bladder cancer, the functional significance of LOH remains to be clarified. The present study introduced human chromosome 9q into basal bladder cancer cell line, SCaBER, using microcell-mediated chromosome transfer to investigate the effect of LOH of 9q on molecular bladder cancer subtypes. These cells demonstrated decreased proliferation and migration capacity compared with parental and control cells. Conversely, transfer of human chromosome 4 did not change the cell phenotype. Expression level of peroxisome proliferator-activated receptor (PPAR)γ, a marker of luminal type, increased 3.0-4.4 fold in SCaBER cells altered with 9q compared with parental SCaBER cells. Furthermore, the expression levels of tumor suppressor PTEN, which regulates PPARγ, also increased in 9q-altered cells. These results suggested that human chromosome 9q may carry regulatory genes for PPARγ that are involved in the progression of neoplastic transformation of bladder cancer.

PITX1 inhibits the growth and proliferation of melanoma cells through regulation of SOX family genes.

Melanoma is one of the most aggressive types of cancer wherein resistance to treatment prevails. Therefore, it is important to discover novel molecular targets of melanoma progression as potential treatments. Here we show that paired-like homeodomain transcription factor 1 (PITX1) plays a crucial role in the inhibition of melanoma progression through regulation of SRY-box transcription factors (SOX) gene family mRNA transcription. Overexpression of PITX1 in melanoma cell lines resulted in a reduction in cell proliferation and an increase in apoptosis. Additionally, analysis of protein levels revealed an antagonistic cross-regulation between SOX9 and SOX10. Interestingly, PITX1 binds to the SOX9 promoter region as a positive regulatory transcription factor; PITX1 mRNA expression levels were positively correlated with SOX9 expression, and negatively correlated with SOX10 expression in melanoma tissues. Furthermore, transcription of the long noncoding RNA (lncRNA), survival-associated mitochondrial melanoma-specific oncogenic noncoding RNA (SAMMSON), was decreased in PITX1-overexpressing cells. Taken together, the findings in this study indicate that PITX1 may act as a negative regulatory factor in the development and progression of melanoma via direct targeting of the SOX signaling.

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.

An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells.

Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.

Possible Relationship Between MYBL1 Alterations and Specific Primary Sites in Adenoid Cystic Carcinoma: A Clinicopathological and Molecular Study of 36 Cases.

BACKGROUND: Adenoid cystic carcinoma (ACC) is a relatively rare malignant neoplasm that occurs in salivary glands and various other organs. Recent studies have revealed that a significant proportion of ACCs harbor gene alterations involving MYB or MYBL1 (mostly fusions with NFIB) in a mutually-exclusive manner. However, its clinical significance remains to be well-established. METHODS: We investigated clinicopathological and molecular features of 36 ACCs with special emphasis on the significance of MYBL1 alterations. Reverse-transcription polymerase-chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) were performed to detect MYB/MYBL1-NFIB fusions and MYBL1 alterations, respectively. Immunohistochemistry was performed to evaluate MYB expression in the tumors. The results were correlated with clinicopathological profiles of the patients. RESULTS: RT-PCR revealed MYB-NFIB and MYBL1-NFIB fusions in 10 (27.8%) and 7 (19.4%) ACCs, respectively, in a mutually-exclusive manner. FISH for MYBL1 rearrangements was successfully performed in 11 cases, and the results were concordant with those of RT-PCR. Immunohistochemically, strong MYB expression was observed in 23 (63.9%) tumors, none of which showed MYBL1 alterations. Clinicopathologically, a trend of a better disease-specific survival was noted in patients with MYBL1 alterations than in those with MYB-NFIB fusions and/or strong MYB expression; however, the difference was not significant. Interestingly, we found tumors with MYBL1 alterations significantly frequently occurred in the mandibular regions (P = 0.012). Moreover, literature review revealed a similar tendency in a previous study. CONCLUSION: Our results suggest that there are some biological or etiological differences between ACCs with MYB and MYBL1 alterations. Moreover, the frequent occurrence of MYBL1-associated ACC in the mandibular regions suggests that MYB immunohistochemistry is less useful in diagnosing ACCs arising in these regions. Further studies are warranted to verify our findings.

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription.

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.

Regulation of functional KCNQ1OT1 lncRNA by ß-catenin.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes through epigenetic mechanisms. We previously reported that KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster. Furthermore, aberration of KCNQ1OT1 transcription was observed with a high frequency in colorectal cancers. However, the molecular mechanism of the transcriptional regulation and the functional role of KCNQ1OT1 in colorectal cancer remain unclear. Here, we show that the KCNQ1OT1 transcriptional level was significantly increased in human colorectal cancer cells in which ß-catenin was excessively accumulated in the nucleus. Additionally, overexpression of ß-catenin resulted in an increase in KCNQ1OT1 lncRNA-coated territory. On the other hand, knockdown of ß-catenin resulted in significant decrease of KCNQ1OT1 lncRNA-coated territory and an increase in the mRNA expression of the SLC22A18 and PHLDA2 genes that are regulated by KCNQ1OT1. We showed that ß-catenin can promote KCNQ1OT1 transcription through direct binding to the KCNQ1OT1 promoter. Our evidence indicates that ß-catenin signaling may contribute to development of colorectal cancer by functioning as a novel lncRNA regulatory factor via direct targeting of KCNQ1OT1.

Studies of Tumor Suppressor Genes via Chromosome Engineering.

The development and progression of malignant tumors likely result from consecutive accumulation of genetic alterations, including dysfunctional tumor suppressor genes. However, the signaling mechanisms that underlie the development of tumors have not yet been completely elucidated. Discovery of novel tumor-related genes plays a crucial role in our understanding of the development and progression of malignant tumors. Chromosome engineering technology based on microcell-mediated chromosome transfer (MMCT) is an effective approach for identification of tumor suppressor genes. The studies have revealed at least five tumor suppression effects. The discovery of novel tumor suppressor genes provide greater understanding of the complex signaling pathways that underlie the development and progression of malignant tumors. These advances are being exploited to develop targeted drugs and new biological therapies for cancer.

miR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells.

Human telomerase reverse transcriptase (hTERT) plays a crucial role in cancer development. We previously identified paired-like homeodomain1 (PITX1) as an hTERT suppressor gene. However, the underlying mechanisms that are involved in the regulation of PITX1 remain unknown. Here, we report that the microRNA-19b (miR-19b) regulates hTERT expression and cell proliferation through inhibition of PITX1. Compared with normal melanocyte cells, miR-19b expression was higher in most melanoma cells and was accompanied by downregulation of PITX1. Moreover, overexpression of miR-19b inhibited PITX1 mRNA translation through a miR-19b binding site within the 3'UTR of the PITX1 mRNA. Our combined findings indicate the participation of miR-19b as a novel upstream effector of hTERT transcription via direct targeting of PITX1.