Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction

Even if template sequence of hTR played an essential role in telomere binding, a 326 nucleotide fragment of hTR containing template, pseudoknot, and CR4–5 domains is critical for both binding with telomeric DNA and reconstitution of telomerase activity. A functional study with antisense oligonucleotides suggested that targeted disruption of the template region efficiently abrogated both telomeric DNA binding and telomerase activity, whereas disruption of the CR4–5 region induced only loss of telomerase activity. hTR interacts with telomeric DNA via structural region composed of the template, pseudoknot, and CR4–5 domains, however, each structural domain plays a distinct role in telomere binding and telomerase activity reconstitution.ope

In vivo selection of metastatic clones of gastric cancer cell line by orthotopic implantation model

의과학과/박사[한글] 위암의 간전이 기전연구를 위하여 위암환자의 혈액에서 분리하여 수립한 YCC-16 세포주로부터 위암 동소이식 모델을 확립한다음 간전이 세포주 클론들을 계대동소이식을 통하여 확보하였다. 확립된 클론들의 생물학적 특성은 세포성장곡선 측정과 이동능 측정을 위한 Boyden chamber assay, 집락형성능 측정을 위한 soft agar assay, 그리고 세포침윤능 조사를 위한 zymography를 시행하여 조사하였다. 또한 유전적 특성은 핵형분석, cDNA microarray-based comparative genomic hybridization (micro-CGH)과 cDNA microarray로 조사하여 다음과 같은 결과를 얻었다. 1. 동소이식 동물모델에서 모세포주인 YCC-16은 간, 폐, 비장 그리고 복강내 림프절로의 광범한 전이 양상을 보이는데 비해 (3마리/3마리, 100%), 간전이에서 얻어진 클론들은 간으로만 전이하였다. 또한 계대동소이식을 거친 전이클론들의 간전이 비율은 향상되었으나 (passageⅡ: 2마리/5마리, 40%; passageⅢ: 3마리/5마리, 60%; passageⅣ: 3마리/5마리, 60%) 세포형태는 큰 차이를 보이지 않았다. 2. 모세포주인 YCC-16에 비해 동소이식 원발부위 세포주인 S1L0은 이동능과 클론형성능이 감소하였다 (YCC-16 vs S1L0, p=0.045). 반면 간전이 클론들에서는 이동능이 증가되었으며 (S1L0 vs S1L1, S2L2와 S3L3, p=0.045), 침윤능 및 클론형성능은 계대동소이식에 따라 증가하였다 (S1L1 vs S2L2 vs S3L3, p=0.045). 3. 모세포주인 YCC-16과 동소이식 원발부위인 위에서 수립한 종양세포주 S1L0, 간전이 계대동소이식별로 수립한 간전이 세포주클론들인 S1L1, S2L2와 S3L3에서 유전적 특성을 비교하기 위하여 핵형분석과 micro-CGH를 시행한 결과, 각 클론들의 유전적 배경은 동일하지만 클론별로는 서로 다른 유전자 변화를 나타내었다. 각 클론들의 핵형과 micro-CGH 결과를 비교분석하여 40개의 유전자들을 발굴하였고, 이들 중 mRNA 발현변화를 보이는 10개 유전자들을 선별하였다. 선별된 유전자들은 세포의 증식, 분화와 신호전달에 관여하는 유전자들임을 확인하였다. 또한 모세포주인 YCC-16과 간전이 클론들을 cDNA microarray를 통하여 비교한 결과 chemokine인 CXCL1 등 23개의 간전이 관련 유전자들을 선별하였다. 다섯가지 세포주에서 세포부착능과 관련되어 발현차이를 나타내는 유전자 31개를 발굴하였으며, 그 중에서 dystroglycan (DAG1)은 동소이식 원발부위 종양세포주인 S1L0에서 발현이 가장 많았고 간전이 클론들에서는 계대동소이식에 따라서 증가하는 양상을 보였다. 추가적으로 간전이 클론들에서 계대동소이식별로의 간전이능이 증가함에 따라 발현이 단계적으로 증가 또는 감소를 보이는 4개의 유전자들을 선별하였으며 (transmembrane protein 16A, RAC CDC42 exchange factor, UNC-5 homolog C, EST), 이들은 세포의 증식과 신호전달에 관여하는 유전자들임을 확인하였다. 모세포주인 YCC-16과 간전이 클론들과의 유전적 변화의 차이는 주로 부착능 등 분자들의 발현차이로 나타났으며, 특히 간전이 클론들에서 세포의 증식과 신호전달에 관여하는 분자들의 발현차이를 확인하였다. 결론적으로 이런 유전적 특성의 변화는 생물학적 특성의 변화로 표현되어 간전이 클론들에서는 계대동소이식에 따라 침윤성과 클론형성능이 향상되었으며, 궁극적으로 전이능이 증가되었다. [영문]We established liver metastatic subclones of YCC-16 using repeated orthotopic implantation in nude mice, named as S1L1, S2L2, and S3L3. We carried out biologic and genetic evaluations to characterize and compare the established cell lines. The results were as follows; 1. Parental cell line, YCC-16, showed wide-spread metastasis over liver, lung, spleen and intra-abdominal lymph nodes (3/3, 100%), but 3 metastatic subclones were characterized by liver-metastatic patterns only. The cellular morphologies appeared no differences with one another, though metastatic tendency was increasing as passages proceeded (passageⅡ: 2/5, 40%; passageⅢ: 3/5, 60%; passageⅣ: 3/5, 60%). 2. Metastatic subclones showed the increasing tendency in clonogenecity and the invasiveness following the passages (S1L1 vs S2L2 vs S3L3, p=0.045) and the increasing motility compared with the orthotopic primary cell line (S1L0 vs S1L1, S2L2, S3L3, p=0.045). This indicated that cells with increased clonogenecity might be prone to metastasize. 3. Each subclone had its own distinct karyotypic pattern despite of similar background in general. 40 genes with genetic aberrations were screened by comparing karyotype and micro-CGH between patental cell line and the subclones. Furthermore, 10 genes were selected by matched to the genes expression levels. We found that the genes were mostly involved in cellular proliferation, differentiation and signal transduction. Twenty three genes were screened by comparing YCC-16 and liver-metastatic subclones using cDNA microarray, which might be related to liver specific metastasis. Among the adhesion related genes, 31 genes showing differential expressions among the YCC-16, orthotopic primary S1L0 and the liver metastatic subclones were selected. The result of semiquantitative real-time PCR of DAG1, producing adhesion-related dystroglycan, showed that the expression levels of DAG1 in YCC-16 and metastatic subclones were lower than that of orthotopic primary S1L0. In addition 4 genes were selected, which showed stepwise patterns of increasing or decreasing expressions in the 3 liver-metastatic subclones (transmembrane protein 16A, RAC/CDC42 exchange factor, UNC-5 homolog C, EST). They were generally involved in cell proliferation and signal transduction. In conclusion, the divergences of YCC-16 and the metastatic clones in genetic change was shown in differential expression of adhesion-related molecules. The major genetic changes characterizing liver-metastatic subclones were mainly attributed to the differential expressions of proliferation and signal transduction related genes and we observed that the genetic changes caused cellular phenotypic changes, resulting in a metastatic process.restrictio

Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells

Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.restrictio