Structural and Mass Spectrometric Imaging Analyses of Adhered Tunic and Adhesive Projections of Solitary Ascidians.

Most ascidian species settle on underwater substrates during a short free-swimming tadpole larval period. During this process, "rapid adhesion" occurs on adhesive papillae located at the anterior region of the cephalenteron. Settled and transformed ascidians subsequently expand the attachment area by "slow adhesion" with ampullae. In the present study, we attempted to identify the ultrastructures related to the adhesion process and adhesive materials in the ascidian tunic and to elucidate the biological function of vanadium in adhesion. We focused on an adhesive organ named the adhesive projection, which is newly generated by the adhered tunic to enlarge the bonding area between ascidian and substrate. Based on its structure and the presence of vanadiumcontaining blood cells, the adhesive projection was considered to be a large tunic vessel. At the adhered tunic, eosinophilic regions and migrated tunic cells were observed, but metal deposition was not detected. We speculate that the eosinophilic materials were components of the adhesive glue, and these are likey produced in epithelial cells, tunic cells, or both. Furthermore, using imaging mass spectrometry, we identified eight tunic-specific molecules as glue candidates.

Detection of MYCN amplification using blood plasma: noninvasive therapy evaluation and prediction of prognosis in neuroblastoma.

PURPOSE: Amplification of neuroblastoma derived (avian)v-myc myelocytomatosis viral related oncogene (MYCN) is an important risk-stratified indicator in neuroblastoma. To evaluate the feasibility of noninvasive measurement of MYCN amplification, we analyzed MYCN amplification in stored blood plasma samples. METHODS: We used quantitative real-time PCR to determine MYCN copy numbers in plasma-derived DNA of 10 healthy volunteers and 50 neuroblastoma cases. The copy number was calculated as the ratio of copies of MYCN to those of a reference gene. Plasma samples obtained after surgery or neoadjuvant therapy were also analyzed in five cases and four cases, respectively. RESULTS: In 34 neuroblastoma cases, MYCN was nonamplified in both tumor tissue and blood plasma. In 16 neuroblastoma cases, MYCN was amplified in both tumor tissue and blood plasma; 13 of the 16 cases showed poor outcomes. MYCN amplification was undetectable in blood plasma shortly after surgery or neoadjuvant therapy. The correlation coefficient between MYCN copy numbers in tumor tissue and in blood plasma was approximately 0.9. CONCLUSION: We can detect MYCN amplification of tumor tissue noninvasively and quantitatively by measuring the MYCN copy number in blood plasma. Determination of MYCN copy number in plasma may be useful when evaluating surgery and neoadjuvant chemotherapy.

Neoplastic transformation by TERT in FGF-2-expanded human mesenchymal stem cells.

The low percentage of human mesenchymal stem cells (hMSCs) in bone marrow necessitates their in vitro expansion prior to clinical use in regenerative medicine. We evaluated the effect of long-term culture of hMSCs on telomere length and transformation capacity by TERT transfection. hMSCs were isolated from the bone marrow aspirates of 24 donors and cultured with fibroblast growth factor-2 (FGF-2). Six cell lines with >500 population doubling levels were considered immortalized. TERT was transfected into two of the six lines for a comparison of telomere length, telomerase activity, differential capacity, colony formation capacity in soft agar and tumorigenicity in immunodeficient (NOD-SCID) mice. hMSC lines exhibited elongated telomeres without the activation of telomerase and retained multi-lineage differentiation potential upon chondrogenic or adipogenic differentiation, while non-immortalized hMSCs showed a marked reduction in telomere length in the differentiation process. Immortalized hMSCs showed anchorage-independence and formed tumors in NOD-SCID mice. Histologically, these tumors consisted of differentiated cells such as fat tissue and cartilage. Two TERT-transfected hMSC lines showed high rates of tumor formation in NOD-SCID mice. These tumors were histologically similar to teratocarcinoma without differentiated cells. These cells may provide a model for the origin of cancer stem cells from adult stem cells, and indicate the possibility that telomerase activation has a major role in the malignant transformation of human stem cells. These data suggest that adult hMSCs have a potential for neoplastic transformation and have implications for the use of hMSCs in tissue engineering and regenerative medicine.

Telomerase activation without shortening of telomeric 3'-overhang is a poor prognostic factor in human colorectal cancer.

Our previous report demonstrated a good correlation between high telomerase activity of cancer tissues and a poor prognosis of patients with colorectal cancers, except for several cases. To elucidate the additional factors that contribute to patient prognosis, the correlation among the expression levels of telomere binding proteins (TBP), the lengths of telomeres, the lengths of telomere 3'-overhang (3'-OH) and telomerase activity in 106 paired colorectal cancer and corresponding noncancerous mucosa (NCM) specimens were examined. The expression levels of eight TBP genes (TRF1, TRF2, TIN2, TANK1, TANK2, POT1, RAP1 and TPP1) were analyzed. Among the 106 cases, 35 cases had shortened telomeres (<7 kb), 15 had shortened 3'-OH (3'-OH length ratio of cancer/NCM <0.5) and 88 were classified as telomerase-activated cancers (activity ratio of cancer/NCM >2). Comparison between NCM and cancer in each case showed that all TBP except for POT1 were downregulated in cancers. A survival analysis using a Cox proportional hazard model showed that the survival rate of the telomerase-activated cases with shortened 3'-OH and that of telomerase-inactivated cases were significantly better than that of telomerase-activated cases without 3'-OH shortening, that is, restored or maintained 3'-OH (P = 0.018). In the telomerase-activated cancers, the length of 3'-OH was significantly correlated with the expression levels of POT1. Elongation of telomeric overhang by telomerase, which might be regulated by POT1, may contribute to the increase of malignant potential in colorectal cancers.

Detection of CpG island hypermethylation of caspase-8 in neuroblastoma using an oligonucleotide array.

BACKGROUND: The caspase-8 gene (CASP8) is frequently inactivated in unfavorable neuroblastomas through DNA methylation. The present study utilized oligoarrays to evaluate the methylation status of a CpG island located between exons 2 and 3 of caspase 8 in neuroblastomas. PROCEDURE: DNA derived from 70 neuroblastomas was amplified by PCR after bisulfate modification and subjected to analysis on a self-made oligoarray that utilized a polycarbodiimide-coated slide to detect methylation of six intragenic CpG islands of caspase 8. In 30 cases, the methylation status was also analyzed by sequencing. In six cases, the PCR product was cloned into a vector and analyzed. RESULTS: Among the 70 tumor-derived DNAs, methylation was not detected in 18 cases, one methylated CpG was found in 12 cases, two in 18 cases, three in 3 cases, four in 8 cases, five in 1 case and six in 10 cases. All methylated CpG loci detected by sequencing were detected by oligoarray, but some methylated CpGs in three loci were detected by oligoarray alone. In these discrepant loci, methylation was detected in some clones after subcloning, indicating that the oligoarray might be more sensitive than sequencing. The CASP8 expression level was depressed in the tumors having two distinct CpG doublets. These results were significantly correlated with MYCN amplification and with clinical outcomes. CONCLUSIONS: A significant difference in the methylation status within the CpG island of CASP8 was shown between favorable and unfavorable subtypes, and CASP8 methylation detected by oligoarray may be useful in the clinical evaluation of neuroblastomas.

Exploration of the genes responsible for unlimited proliferation of immortalized lung fibroblasts.

Regulation mechanism of lung fibroblast proliferation remains unknown. To elucidate the key molecules in it, the authors here established mortal and immortal nontransformed lung fibroblast cell line/strains with elongated life span by telomerase reverse transcriptase gene transfection. Comparing the expression profiles of them, 51 genes were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. This set of fibrobrast strains of same origin with different proliferative capacities may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide biomarkers or therapeutic targets of pulmonary fibrosis.